Methods and compositions for needleless delivery of macromolecules

ABSTRACT

Methods and compositions for needleless delivery of macromolecules to the bloodstream of a subject are provided herein. In one aspect, the invention provides a delivery construct, comprising a receptor binding domain, a transcytosis domain, a macromolecule to be delivered to a subject, and a cleavable linker. Generally, the cleavable linker is cleavable by an enzyme present in higher concentration at or near the basal-lateral membrane of a polarized epithelial cell or in the plasma than elsewhere in the body, for example, at the apical side of the polarized epithelial cell. In other aspects, the invention provides nucleic acids encoding delivery constructs of the invention, kits comprising delivery constructs of the invention, cells expressing delivery constructs of the invention, and methods of using delivery constructs of the invention.

This application is entitled to and claims benefit of U.S. ProvisionalApplication No. 60/615,970, filed Oct. 4, 2004, of U.S. ProvisionalApplication No. 60/684,484, filed May 24, 2005, and of U.S. ProvisionalApplication No. 60/718,907, filed Sep. 19, 2005, each of which is herebyincorporated by reference in its entirety.

1. FIELD OF THE INVENTION

The present invention relates, in part, to methods and compositions forneedleless delivery of macromolecules to a subject. In one aspect, themethods and compositions involve administering to the subject a deliveryconstruct comprising the macromolecule to be delivered, wherein themacromolecule is linked to the remainder of the construct with a linkerthat is cleavable at a basal-lateral membrane of a polarized epithelialcell.

2. BACKGROUND

Advances in biochemistry and molecular biology have resultedidentification and characterization of many therapeutic macromolecules,including, for example, growth hormone, erythropoietin, insulin, IGF,and the like. Administration of these molecules can result in drasticimprovements in quality of life for subjects afflict with a wide rangeof ailments.

However, administration of these therapeutic macromolecules remainsproblematic. Currently, therapeutic macromolecules are typicallyadministered by injection. Such injections require penetration of thesubject's skin and tissues and are associated with pain. Further,penetration of the skin breaches one effective nonspecific mechanism ofprotection against infection, and thus can lead to potentially seriousinfection.

Previous attempts have been made for needleless delivery ofmacromolecules to subjects. See, e.g., International Patent PublicationNo. WO 01/30,392. In these efforts, delivery vehicles are used todeliver macromolecules to a subject through polarized epithelial cells.However, these derivatives lack a cleavable linker that is cleavable byan enzyme at the basal-lateral membrane of a polarized epithelial cell.Without cleavage, the probability of induction of an immune responseagainst the delivery vehicle and/or the macromolecule to be delivered isincreased. Also, steric hindrance between the delivery vehicle and themacromolecule can reduce the activity of the macromolecule, reducingefficacy the treatment.

Accordingly, there is an unmet need for new methods and compositionsthat can be used to administer macromolecules to subjects withoutbreaching the skin of the subject. This and other needs are met by themethods and compositions of the present invention.

3. SUMMARY OF THE INVENTION

The delivery constructs of the invention comprise a macromolecule fordelivery to a subject that is linked to the remainder of the constructwith a cleavable linker. The linker is cleavable by an enzyme or anenvironmental cue that is present at the basal-lateral membrane of anepithelial cell.

Accordingly, in certain aspects, the invention provides a deliveryconstruct comprising a receptor binding domain, a transcytosis domain, amacromolecule to be delivered to a subject, and a cleavable linker.Cleavage at the cleavable linker can separate the macromolecule from theremainder of the delivery construct. In certain embodiments, thecleavable linker can be cleavable by an enzyme that is present at abasal-lateral membrane of a polarized epithelial cell of the subject. Inother embodiments, the cleavable linker can be cleavable by an enzymethat is present in the plasma of said subject.

In another aspect, the invention provides a polynucleotide that encodesa delivery construct comprising a receptor binding domain, atranscytosis domain, a macromolecule to be delivered to a subject, and acleavable linker. Cleavage at the cleavable linker can separate themacromolecule from the remainder of the delivery construct. In certainembodiments, the cleavable linker can be cleavable by an enzyme that ispresent at a basal-lateral membrane of a polarized epithelial cell ofthe subject. In other embodiments, the cleavable linker can be cleavableby an enzyme that is present in the plasma of said subject.

In other embodiments, the polynucleotide that encodes a deliveryconstruct comprises a nucleic acid sequence encoding a receptor bindingdomain, a nucleic acid sequence encoding a transcytosis domain, anucleic acid sequence encoding a cleavable linker, and a nucleic acidsequence comprising a polylinker insertion site. The polylinkerinsertion site can be oriented relative to the nucleic acid sequenceencoding a cleavable linker to allow to cleavage of the cleavable linkerto separate a macromolecule that is encoded by a nucleic acid insertedinto the polylinker insertion site from the remainder of the encodeddelivery construct. In certain embodiments, the cleavable linker can becleavable by an enzyme that is present at a basal-lateral membrane of apolarized epithelial cell of the subject. In other embodiments, thecleavable linker can be cleavable by an enzyme that is present in theplasma of said subject.

In another aspect, the invention provides an expression vectorcomprising a polynucleotide of the invention.

In still another aspect, the invention provides a cell comprising anexpression vector of the invention.

In yet another aspect, the invention provides a composition comprising adelivery construct of the invention. In certain embodiments, thecomposition is a pharmaceutical composition.

In still another aspect, the invention provides a method for deliveringa macromolecule to a subject. The method comprises contacting an apicalsurface of a polarized epithelial cell of the subject with a deliveryconstruct of the invention. The delivery construct can comprise areceptor binding domain, a transcytosis domain, a cleavable linker, andthe macromolecule to be delivered. The transcytosis domain cantranscytose the macromolecule to and through the basal-lateral membraneof the epithelial cell. In certain embodiments, the cleavable linker canbe cleaved by an enzyme that is present at a basal-lateral membrane of apolarized epithelial cell of the subject. In other embodiments, thecleavable linker can be cleavable by an enzyme that is present in theplasma of the subject. Cleavage at the cleavable linker can separate themacromolecule from the remainder of the delivery construct, and candeliver the macromolecule to the subject free from the remainder of theconstruct.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D present micrographs showing adhesion to and transport acrossmouse tracheal epithelium of an exemplary delivery construct comprisinggreen fluorescent protein (GFP) (Panels A-C), while GFP alone does notadhere to the epithelial cells (Panel D).

FIG. 2 presents a time course of serum nt-PE-GFP concentrationsfollowing intranasal administration of 100 μg nt-PE-GFP to anesthetizedmice.

FIG. 3 presents the amino acid sequence of an exemplary PE.

FIG. 4 presents a western blot snowing transport and cleavage of anexemplary delivery construct comprising rat growth hormone (rGH) byhuman intestinal epithelial cell monolayers. The delivery construct wasincubated in contact with the apical side of the epithelial cellmonolayer for 4 hours. Media isolated from the basolateral side of themembrane post-incubation (lane 1) contained rGH of native apparentmolecular weight, while media from the apical side of the membrane (lane2) contained intact delivery construct. Lanes 3 and 4 show intactDelivery Construct 2 and recombinant rGH, respectively.

FIG. 5 presents serum rGH concentrations in BALB/c mice which were dosedby subcutaneous (SC) injection of 30 μg of non-glycosylated recombinantrat GH. Individual mice sera were tested at a dilution of 1:10, and thegroup average of rGH concentration were reported (n=4 mice per timepoint). Standard error of the mean (SEM) was indicated by the errorbars.

FIG. 6 presents serum rGH concentrations in BALB/c mice which were dosedorally with 100 μg of Delivery Construct 2. Individual mice sera weretested at a dilution of 1:10, and the group average of rGH concentrationwere reported (n=4 mice per time point). Standard error of the mean(SEM) was indicated by the error bars.

FIG. 7 presents a graphical representation comparing pharmacokinetics ofrGH delivered subcutaneously and with Delivery Construct 2.

FIG. 8 shows expression levels of IGF-1-BP3 mRNA in the liver of femaleBALB/c mice treated with 30 μg recombinant rGH by subcutaneous injectionor with 100 μg of Delivery Construct 2 by oral gavage. Total RNAextracted from the liver was subjected to quantitative RT-PCR usingprimers specific for IGF-1-BP3, as described above. Values werenormalized to glyceraldehyde-3 phosphate dehydrogenase (GAPDH) andexpressed as % of control.

FIG. 9 shows expression levels of growth hormone (GH) receptor mRNA inthe liver of female BALB/c mice treated with rGH by subcutaneousinjection (30 μg) or Delivery Construct 2 by oral gavage (100 μg). TotalRNA extracted from the liver was subjected to quantitative RT-PCR usingprimers specific for GH receptor, shown above. Values were normalized toglyceraldehyde-3 phosphate dehydrogenase (GAPDH) and expressed as % ofcontrol.

FIG. 10 snows expression levels or insulin-like growth factor I (TGF-I);mRNA in the liver of female BALB/c mice treated with rGH by subcutaneousinjection (30 μg) or Delivery Construct 2 by oral gavage (100 μg). TotalRNA extracted from the liver was subjected to quantitative RT-PCR usingprimers specific for IGF-I, shown above. Values were normalized toglyceraldehyde-3 phosphate dehydrogenase (GAPDH) and expressed as % ofcontrol.

FIGS. 11A and B show serum anti-rGH IgG antibodies (diluted 1:25 in FIG.11A and 1:200 in FIG. 11B) of mice orally administered 3,10, or 30 μgDelivery Construct 2 (F2) or subcutaneously administered 3 or 10 μg rGHin graphs with error bars, while FIGS. 11C and D presents the same datafrom individual animals.

FIG. 12 shows a nucleotide sequence that encodes Delivery Construct 6(SEQ ID NO:34), an exemplary Delivery Construct for delivering humangrowth hormone (hGH).

FIGS. 13A and B show the amino acid sequence of Delivery Construct 6(SEQ ID NO:35), an exemplary Delivery Construct for delivering hGH.

FIG. 14 shows a nucleotide sequence that encodes Delivery Construct 7(SEQ ID NO:36), an exemplary Delivery Construct for deliveringinterferon-α (IFN-α).

FIGS. 15A and B show the amino acid sequence of Delivery Construct 7(SEQ ID NO:37), an exemplary Delivery Construct for delivering IFN-α.

FIG. 16 shows 6 presents serum IFN-α concentrations in BALB/c mice whichwere dosed orally with 100 μg of Delivery Construct 8. Individual micesera were tested at a dilution of 1:10, and the group average of IFN-αconcentration were reported (n=3 mice per time point).

FIGS. 17A and B show the amino acid sequence of Delivery Construct 8(SEQ ID NO:38), an exemplary Delivery Construct for deliveringproinsulin.

FIGS. 18 A and B shows the amino acid sequence of the two amino acidchains of Delivery Construct 9 (SEQ ID NOs:39 and 40, respectively), anexemplary Delivery Construct for delivering insulin. Disulfide bonds areformed between the cysteine at position 7 of SEQ ID NO:40 (shown in FIG.18B) and the cysteine at position 381 of SEQ ID NO:39 (shown in FIG.18A) and between the cysteine at position 20 of SEQ ID NO:40 and thecysteine at position 393 of SEQ ID NO:39.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

A “ligand” is a compound that specifically binds to a target molecule.Exemplary ligands include, but are not limited to, an antibody, acytokine, a substrate, a signaling molecule, and the like.

A “receptor” is compound that specifically binds to a ligand.

A ligand or a receptor (e.g., an antibody) “specifically binds to” or“is specifically immunoreactive with” another molecule when the ligandor receptor functions in a binding reaction that indicates the presenceof the molecule in a sample of heterogeneous compounds. Thus, underdesignated assay (e.g., immunoassay) conditions, the ligand or receptorbinds preferentially to a particular compound and does not bind in asignificant amount to other compounds present in the sample. Forexample, a polynucleotide specifically binds under hybridizationconditions to another polynucleotide comprising a complementary sequenceand an antibody specifically binds under immunoassay conditions to anantigen bearing an epitope used to induce the antibody.

“Immunoassay” refers to a method of detecting an analyte in a sampleinvolving contacting the sample with an antibody that specifically bindsto the analyte and detecting binding between the antibody and theanalyte. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York, for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.In one example, an antibody that binds a particular antigen with anaffinity (K_(m)) of about 10 μM specifically binds the antigen.

“Linker” refers to a molecule that joins two other molecules, eithercovalently, or through ionic, van der Waals or hydrogen bonds, e.g., anucleic acid molecule that hybridizes to one complementary sequence atthe 5′ end and to another complementary sequence at the 3′ end, thusjoining two non-complementary sequences. A “cleavable linker” refers toa linker that can be degraded or otherwise severed to separate the twocomponents connected by the cleavable linker. Cleavable linkers aregenerally cleaved by enzymes, typically peptidases, proteases,nucleases, lipases, and the like. Cleavable linkers may also be cleavedby environmental cues, such as, for example, changes in temperature, pH,salt concentration, etc. when there is such a change in environmentfollowing transcytosis of the delivery construct across a polarizedepithelial membrane.

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in an animal. A pharmaceutical composition comprisesa pharmacologically effective amount of an active agent and apharmaceutically acceptable carrier. “Pharmacologically effectiveamount” refers to that amount of an agent effective to produce theintended pharmacological result. “Pharmaceutically acceptable carrier”refers to any of the standard pharmaceutical carriers, vehicles,buffers, and excipients, such as a phosphate buffered saline solution,5% aqueous solution of dextrose, and emulsions, such as an oil/water orwater/oil emulsion, and various types of wetting agents and/oradjuvants. Suitable pharmaceutical carriers and formulations aredescribed in Remington's Pharmaceutical Sciences, 21st Ed. 2005, MackPublishing Co., Easton. A “pharmaceutically acceptable salt” is a saltthat can be formulated into a compound for pharmaceutical use including,e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) andsalts of ammonia or organic amines.

Preferred pharmaceutical carriers depend upon the intended mode ofadministration of the active agent. Typical modes of administrationinclude enteral (e.g., oral, intranasal, rectal, Or vaginal) orparenteral (e.g., subcutaneous, intramuscular, intravenous orintraperitoneal injection; or topical (e.g., transdermal, ortransmucosal administration).

“Small organic molecule” refers to organic molecules of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes organic biopolymers (e.g., proteins, nucleic acids,etc.). Preferred small organic molecules range in size up to about 5000Da, up to about 2000 Da, or up to about 1000 Da.

A “subject” of diagnosis, treatment, or administration is a human ornon-human animal, including a mammal or a primate, and preferably ahuman.

“Treatment” refers to prophylactic treatment or therapeutic treatment. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology. A “therapeutic”treatment is a treatment administered to a subject who exhibits signs ofpathology for the purpose of diminishing or eliminating those signs.

“Pseudomonas exotoxin A” or “PE” is secreted by Pseudomonas aeruginosaas a 67 kD protein composed of three prominent globular domains (Ia, II,and III) and one small subdomain (Ib) that connects domains II and III.See A. S. Allured et al., 1986, Proc. Natl. Acad. Set 83:1320-1324.Without intending to be bound to any particular theory or mechanism ofaction, domain Ia of PE is believed to mediate cell binding becausedomain Ia specifically binds to the low density lipoproteinreceptor-related protein (“LRP”), also known as the α2-macroglobulinreceptor (“α2-MR”) and CD-91. See M. Z. Kounnas et al., 1992, J. Biol.Chem. 267:12420-23. Domain Ia spans amino acids 1-252. Domain II of PEis believed to mediate transcytosis to the interior of a cell followingbinding of domain Ia to the α2-MR. Domain II spans amino acids 253-364.Certain portions of this domain may be required for secretion of PE fromPseudomonas aeruginosa after its synthesis. See, e.g., Vouloux et al.,2000, J. Bacterol. 182:4051-8. Domain Ib has no known function and spansamino acids 365-399. Domain III mediates cytotoxicity of PE and includesan endoplasmic reticulum retention sequence. PE cytotoxicity is believedto result from ADP ribosylation of elongation factor 2, whichinactivates protein synthesis. Domain III spans amino acids 400-613 ofPE. Deleting amino acid E553 (“ΔE553”) from domain III eliminates EF2ADP ribosylation activity and detoxifies PE. PE having the mutationΔE553 is referred to herein as “PEΔE553.” Genetically modified forms ofPE are described in, e.g., U.S. Pat. Nos. 5,602,095; 5,512,658 and5,458,878 Pseudomonas exotoxin, as used herein, also includesgenetically modified, allelic, and chemically inactivated forms of PEwithin this definition. See, e.g., Vasil et al., 1986, Infect. Immunol.52:538-48. Further, reference to the various domains of PE is madeherein to the reference PE sequence presented as FIG. 3. However, one ormore domain from modified PE, e.g., genetically or chemically modifiedPE, or a portion of such domains, can also be used in the chimericimmunogens of the invention so long as the domains retain functionalactivity. One of skill in the art can readily identify such domains ofsuch modified PE based on, for example, homology to the PE sequenceexemplified in FIG. 3 and test for functional activity using, forexample, the assays described below.

“Polynucleotide” refers to a polymer composed of nucleotide units.Polynucleotides include naturally occurring nucleic acids, such asdeoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. Nucleic acid analogs include those which includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor which include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand having the same sequence as an mRNAtranscribed from that DNA and which are located 5′ to the 5′-end of theRNA transcript are referred to as “upstream sequences”; sequences on theDNA strand having the same sequence as the RNA and which are 3′ to the3′ end of the coding RNA transcript are referred to as “downstreamsequences.”

“Complementary” refers to the topological compatibility or matchingtogether of interacting surfaces of two polynucleotides. Thus, the twomolecules can be described as complementary, and furthermore, thecontact surface characteristics are complementary to each other. A firstpolynucleotide is complementary to a second polynucleotide if thenucleotide sequence of the first polynucleotide is substantiallyidentical to the nucleotide sequence of the polynucleotide bindingpartner of the second polynucleotide, or if the first polynucleotide canhybridize to the second polynucleotide under stringent hybridizationconditions. Thus, the polynucleotide whose sequence 5′-TATAC-3′ iscomplementary to a polynucleotide whose sequence is 5′-GTATA-3′.

The term “% sequence identity” is used interchangeably herein with theterm “% identity” and refers to the level of amino acid sequenceidentity between two or more peptide sequences or the level ofnucleotide sequence identity between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% identity means the same thing as 80% sequence identitydetermined by a defined algorithm, and means that a given sequence is atleast 80% identical to another length of another sequence. Exemplarylevels of sequence identity include, but are not limited to, 60, 70, 80,85, 90, 95, 98% or more sequence identity to a given sequence.

The term “% sequence homology” is used interchangeably herein with theterm “% homology” and refers to the level of amino acid sequencehomology between two or more peptide sequences or the level ofnucleotide sequence homology between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% homology means the same thing as 80% sequence homologydetermined by a defined algorithm, and accordingly a homologue of agiven sequence has greater than 80% sequence homology over a length ofthe given sequence. Exemplary levels of sequence homology include, butare not limited to, 60, 70, 80, 85, 90, 95, 98% or more sequencehomology to a given sequence.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at the NCBI website. See alsoAltschul et al., 1990, J. Mol. Biol. 215:403-10 (with special referenceto the published default setting, i.e., parameters w=4, t=17) andAltschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Sequencesearches are typically carried out using the BLASTP program whenevaluating a given amino acid sequence relative to amino acid sequencesin the GenBank Protein Sequences and other public databases. The BLASTXprogram is preferred for searching nucleic acid sequences that have beentranslated in all reading frames against amino acid sequences in theGenBank Protein Sequences and other public databases. Both BLASTP andBLASTX are run using default parameters of an open gap penalty of 11.0,and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.See id.

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has at least onebond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q) Ser (S) and Thr (T).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds in whichthe pair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded nonpolar amino acids include Ala (A), Gly (G), He(I), Leu (L), Met (M) and Val (V).

“Hydrophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include Arg(R), Asn (N), Asp (D), Glu (E), Gln (Q), His (H), Lys (K), Ser (S) andThr (T).

“Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg et al., 1984, J Mol. Biol.179:125-142. Genetically encoded hydrophobic amino acids include Ala(A), Gly (G), He (I), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), Tyr(Y) and Val (V).

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Asp (D) andGlu (E).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith a hydrogen ion. Genetically encoded basic amino acids include Arg(R), His (H) and Lys (K).

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Amplification” refers to any means by which a polynucleotide sequenceis copied and thus expanded into a larger number of polynucleotidemolecules, e.g., by reverse transcription, polymerase chain reaction,ligase chain reaction, and the like.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

“Probe,” when used in reference to a polynucleotide, refers to apolynucleotide that is capable of specifically hybridizing to adesignated sequence of another polynucleotide. A probe specificallyhybridizes to a target complementary polynucleotide, but need notreflect the exact complementary sequence of the template. In such acase, specific hybridization of the probe to the target depends on thestringency of the hybridization conditions. Probes can be labeled with,e.g., chromogenic, radioactive, or fluorescent moieties and used asdetectable moieties. In instances where a probe provides a point ofinitiation for synthesis of a complementary polynucleotide, a probe canalso be a primer.

“Hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refers to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids can be found in Tijssen, 1993, Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays”, Elsevier, N.Y.; Sambrook etal., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, 3^(rd) ed., NY; and Ausubel et al., eds., Current Edition,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, NY.

Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the Tm for a particular probe.

One example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than about 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes. See Sambrook et al. for adescription of SSC buffer. A high stringency wash can be preceded by alow stringency wash to remove background probe signal. An exemplarymedium stringency wash for a duplex of, e.g., more than about 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An exemplary lowstringency wash for a duplex of, e.g., more than about 100 nucleotides,is 4-6×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.Conventional notation is used herein to portray polypeptide sequences;the beginning of a polypeptide sequence is the amino-terminus, while theend of a polypeptide sequence is the carboxyl-terminus.

The term “protein” typically refers to large polypeptides, for example,polypeptides comprising more than about 50 amino acids. The term“protein” can also refer to dimers, trimers, and multimers that comprisemore than one polypeptide.

“Conservative substitution” refers to the substitution in a polypeptideof an amino acid with a functionally similar amino acid. The followingsix groups each contain amino acids that are conservative substitutionsfor one another:

Alanine (A), Serine (S), and Threonine (T)

Aspartic acid (D) and Glutamic acid (E)

Asparagine (N) and Glutamine (Q)

Arginine (R) and Lysine (K)

Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).

The term “about,” as used herein, unless otherwise indicated, refers toa value that is no more than 10% above or below the value being modifiedby the term. For example, the term “about 5 μg/kg” means a range of from4.5 μg/kg to 5.5 μg/kg. As another example, “about 1 hour” means a rangeof from 48 minutes to 72 minutes.

5.2. Delivery Constructs

Generally, the delivery constructs of the present invention arepolypeptides that have structural domains corresponding to domains Iaand II of PE. These structural domains perform certain functions,including, but not limited to, cell recognition and transcytosis, thatcorrespond to the functions of the domains of PE.

In addition to the portions of the molecule that correspond to PEfunctional domains, the delivery constructs of this invention canfurther comprise a macromolecule for delivery to a biologicalcompartment of a subject. The macromolecule can be introduced into anyportion of the delivery construct that does not disrupt a cell-bindingor transcytosis activity. The macromolecule is connected with theremainder of the delivery construct with a cleavable linker.

Accordingly, the delivery constructs of the invention generally comprisethe following structural elements, each element imparting particularfunctions to the delivery construct: (1) a “receptor binding domain”that functions as a ligand for a cell surface receptor and that mediatesbinding of the construct to a cell; (2) a “transcytosis domain” thatmediates transcytosis from a lumen bordering the apical surface of amucous membrane to the basal-lateral side of a mucous membrane; (3) themacromolecule; and (4) a cleavable linker that connects themacromolecule to the remainder of the delivery construct.

The delivery constructs of the invention offer several advantages overconventional techniques for local or systemic delivery of macromoleculesto a subject. Foremost among such advantages is the ability to deliverthe macromolecule without using a needle to puncture the skin of thesubject. Many subjects require repeated, regular doses ofmacromolecules. For example, diabetics must inject insulin several timesper day to control blood sugar concentrations. Such subjects' quality oflife would be greatly improved if the delivery of a macromolecule couldbe accomplished without injection, by avoiding pain or potentialcomplications associated therewith.

Furthermore, many embodiments of the delivery constructs can beconstructed and expressed in recombinant systems. Recombinant technologyallows one to make a delivery construct having an insertion sitedesigned for introduction of any suitable macromolecule. Such insertionsites allow the skilled artisan to quickly and easily produce deliveryconstructs for delivery of new macromolecules, should the need to do soarise.

In addition, connection of the macromolecule to the remainder of thedelivery construct with a linker that is cleaved by an enzyme present ata basal-lateral membrane of an epithelial cell allows the macromoleculeto be liberated from the delivery construct and released from theremainder of the delivery construct soon after transcytosis across theepithelial membrane. Such liberation reduces the probability ofinduction of an immune response against the macromolecule. It alsoallows the macromolecule to interact with its target free from theremainder of the delivery construct.

Other advantages of the delivery constructs of the invention will beapparent to those of skill in the art.

In certain embodiments, the invention provides a delivery construct thatcomprises a receptor binding domain, a transcytosis domain, amacromolecule to be delivered to a subject, and a cleavable linker.Cleavage at the cleavable linker separates the macromolecule from theremainder of the construct. The cleavable linker is cleavable by anenzyme that is present at a basal-lateral membrane of a polarizedepithelial cell of the subject or in the plasma of the subject. Incertain embodiments, the enzyme that is at a basal-lateral membrane of apolarized epithelial cell exhibits higher activity on the basal-lateralside of a polarized epithelial cell than it does on the apical side ofthe polarized epithelial cell. In certain embodiments, the enzyme thatis in the plasma of the subject exhibits higher activity in the plasmathan it does on the apical side of a polarized epithelial cell.

In certain embodiments, the delivery construct further comprises asecond cleavable linker. In certain embodiments, the first and/or thesecond cleavable linker comprises an amino acid sequence that isselected from the group consisting of Ala-Ala-Pro-Phe (SEQ ID NO.:4),Gly-Gly-Phe (SEQ ID NO.:5), Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu(SEQ ID NO.:7), Ala-Ala-Leu (SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9),Val-Gly-Arg (SEQ ID NO.: 10). In certain embodiments, the first and/orthe second cleavable linker comprises an amino acid sequence that isselected from the group consisting of Ala-Ala-Pro-Phe (SEQ ID NO.:4),Gly-Gly-Phe (SEQ ID NO.:5), Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu(SEQ ID NO.:7), Ala-Ala-Leu (SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9),Val-Gly-Arg (SEQ ID NO.: 10) and is cleavable by an enzyme that exhibitshigher activity on the basal-lateral side of a polarized epithelial cellthan it does on the apical side of the polarized epithelial cell. Incertain embodiments, the first and/or the second cleavable linkercomprises an amino acid sequence that is selected from the groupconsisting of Ala-Ala-Pro-Phe (SEQ ID NO.:4), Gly-Gly-Phe (SEQ IDNO.:5), Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu (SEQ ID NO.:7),Ala-Ala-Leu (SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9), Val-Gly-Arg (SEQID NO.: 10) and is cleavable by an enzyme that exhibits higher activityin the plasma than it does on the apical side of a polarized epithelialcell.

In certain embodiments, the enzyme that is present at a basal-lateralmembrane of a polarized epithelial cell is selected from the groupconsisting of Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin AI,Subtilisin AII, Thrombin I, and Urokinase I.

In certain embodiments, the receptor binding domain is selected from thegroup consisting of receptor binding domains from Pseudomonas exotoxinA, cholera toxin, botulinum toxin, diptheria toxin, shiga toxin, orshiga-like toxin; monoclonal antibodies; polyclonal antibodies;single-chain antibodies; TGF α; EGF; IGF-I; IGF-II; IGF-III; IL-1; IL-2;IL-3; IL-6; MIP-1a; MIP-1b; MCAF; and IL-8. In certain embodiments, thereceptor binding domain binds to a cell-surface receptor that isselected from the group consisting of α2-macroglobulin receptor,epidermal growth factor receptor, transferrin receptor, chemokinereceptor, CD25, CD11B, CD11C, CD80, CD86, TNFα receptor, TOLL receptor,M-CSF receptor, GM-CSF receptor, scavenger receptor, and VEGF receptor.In further embodiments, the receptor binding domain of Pseudomonasexotoxin A is Domain Ia of Pseudomonas exotoxin A. In yet furtherembodiments, the receptor binding domain of Pseudomonas exotoxin A hasan amino acid sequence that is SEQ ID NO.:1.

In certain embodiments, the transcytosis domain is selected from thegroup consisting of transcytosis domains from Pseudomonas exotoxin A,botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin,heat-labile E, coli enterotoxin, shiga toxin, and shiga-like toxin. Infurther embodiments, the transcytosis domain is Pseudomonas exotoxin Atranscytosis domain. In still further embodiments, the Pseudomonasexotoxin A transcytosis domain has an amino acid sequence that is SEQ IDNO.:2.

In certain embodiments, the macromolecule is selected from the group ofa nucleic acid, a peptide, a polypeptide, a protein, and a lipid, hifurther embodiments, the polypeptide is selected from the groupconsisting of polypeptide hormones, cytokines, chemokines, growthfactors, and clotting factors. In yet further embodiments, thepolypeptide is selected from the group consisting of IGF-I, IGF-II,IGF-III, EGF, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, IL-1, IL-2, IL-3,IL-6, IL-8, IL-12, EPO, growth hormone, factor VII, vasopressin,calcitonin, parathyroid hormone, luteinizing hormone-releasing factor,tissue plasminogen activators, adrenocorticototropin, enkephalin, andglucagon-like peptide 1. In still further embodiments, the polypeptideis human growth hormone. In other embodiments, the protein is humaninsulin.

In certain embodiments, the delivery constructs further comprise asecond macromolecule that is selected from the group consisting of anucleic acid, a peptide, a polypeptide, a protein, a lipid, and a smallorganic molecule and a second cleavable linker, wherein cleavage at saidsecond cleavable linker separates said second macromolecule from theremainder of said construct. In certain embodiments, the firstmacromolecule is a first polypeptide and said second macromolecule is asecond polypeptide. In certain embodiments, the first polypeptide andthe second polypeptide associate to form a multimer. In certainembodiments, the multimer is a dimer, tetramer, or octamer. In furtherembodiments, the dimer is an antibody.

5.2.1. Receptor Binding Domain

The delivery constructs of the invention generally comprise a receptorbinding domain. The receptor binding domain can be any receptor bindingdomain known to one of skill in the art without limitation to bind to acell surface receptor that is present on the apical membrane of anepithelial cell. Preferably, the receptor binding domain bindsspecifically to the cell surface receptor. The receptor binding domainshould bind to the cell surface receptor with sufficient affinity toallow endocytosis of the delivery construct.

In certain embodiments, the receptor binding domain can comprise apeptide, a polypeptide, a protein, a lipid, a carbohydrate, or a smallorganic molecule, or a combination thereof. Examples of each of thesemolecules that bind to cell surface receptors present on the apicalmembrane of epithelial cells are well known to those of skill in theart. Suitable peptides or polypeptides include, but are not limited to,bacterial toxin receptor binding domains, such as the receptor bindingdomains from PE, cholera toxin, botulinum toxin, diptheria toxin, shigatoxin, shiga-like toxin, etc.; antibodies, including monoclonal,polyclonal, and single-chain antibodies, or derivatives thereof, growthfactors, such as EGF, IGF-I, IGF-II, IGF-III etc.; cytokines, such asIL-1, IL-2, IL-3, IL-6, etc; chemokines, such as MIP-1a, MIP-1b, MCAF,IL-8, etc.; and other ligands, such as CD4, cell adhesion molecules fromthe immunoglobulin superfamily, integrins, ligands specific for the IgAreceptor, etc. See, e.g., Pastane et al., 1992, Annu. Rev. Biochem.61:331-54; and U.S. Pat. Nos. 5,668,255, 5,696,237, 5,863,745,5,965,406, 6,022,950, 6,051,405, 6,251,392, 6,440,419, and 6,488,926.The skilled artisan can select the appropriate receptor binding domainbased upon the expression pattern of the receptor to which the receptorbinding domain binds.

Lipids suitable for receptor binding domains include, but are notlimited to, lipids that themselves bind cell surface receptors, such assphingosine-1-phosphate, lysophosphatidic acid,sphingosylphosphorylcholine, retinoic acid, etc.; lipoproteins such asapolipoprotein E, apolipoprotein A, etc., and glycolipids such aslipopolysaccharide, etc.; glycosphingolipids such asglobotriaosylceramide and galabiosylceramide; and the like.Carbohydrates suitable for receptor binding domains include, but are notlimited to, monosaccharides, disaccharides, and polysaccharides thatcomprise simple sugars such as glucose, fructose, galactose, etc.; andglycoproteins such as mucins, selectins, and the like. Suitable smallorganic molecules for receptor binding domains include, but are notlimited to, vitamins, such as vitamin A, B₁, B₂, B₃, B₆, B₉, B₁₂, C, D,E, and K, amino acids, and other small molecules that are recognizedand/or taken up by receptors present on the apical surface of epithelialcells. U.S. Pat. No. 5,807,832 provides an example of such small organicmolecule receptor binding domains, vitamin B₁₂.

In certain embodiments, the receptor binding domain can bind to areceptor found on an epithelial cell. In further embodiments, thereceptor binding domain can bind to a receptor found on the apicalmembrane of an epithelial cell. The receptor binding domain can bind toany receptor known to be present on the apical membrane of an epithelialcell by one of skill in the art without limitation. For example, thereceptor binding domain can bind to α2-MR, EGFR, or IGFR. An example ofa receptor binding domain that can bind to α2-MR is domain Ia of PE.Accordingly, in certain embodiments, the receptor binding domain isdomain Ia of PE. In other embodiments, the receptor binding domain is aportion of domain Ia of PE that can bind to α2-MR. Exemplary receptorbinding domains that can bind to EGFR include, but are not limited to,EGF and TGFα. Examples of receptor binding domains that can bind to IGFRinclude, but are not limited to, IGF-I, IGF-II, or IGF-III. Thus, incertain embodiments, the receptor binding domain is EGF, IGF-I, IGF-II,or IGF-III. In other embodiments, the receptor binding domain is aportion of EGF, IGF-I, IGF-II, or IGF-III that can bind to the EGF orIGF receptor.

In certain embodiments, the receptor binding domain binds to a receptorthat is highly expressed on the apical membrane of a polarizedepithelial cell but is not expressed or expressed at low levels onantigen presenting cells, such as, for example, dendritic cells.Exemplary receptor binding domains that have this kind of expressionpattern include, but are not limited to, TGFα, EGF, IGF-I, IGF-II, andIGF-III.

In certain embodiments, the delivery constructs of the inventioncomprise more than one domain that can function as a receptor bindingdomain. For example, the delivery construct can comprise PE domain Ia inaddition to another receptor binding domain.

The receptor binding domain can be attached to the remainder of thedelivery construct by any method or means known by one of skill in theart to be useful for attaching such molecules, without limitation. Incertain embodiments, the receptor binding domain is expressed togetherwith the remainder of the delivery construct as a fusion protein. Suchembodiments are particularly useful when the receptor binding domain andthe remainder of the construct are formed from peptides or polypeptides.

In other embodiments, the receptor binding domain is connected with theremainder of the delivery construct with a linker. In yet otherembodiments, the receptor binding domain is connected with the remainderof the delivery construct without a linker. Either of these embodimentsare useful when the receptor binding domain comprises a peptide,polypeptide, protein, lipid, carbohydrate, nucleic acid, or smallorganic molecule.

In certain embodiments, the linker can form a covalent bond between thereceptor binding domain and the remainder of the delivery construct. Incertain embodiments, the covalent bond can be a peptide bond. In otherembodiments, the linker can link the receptor binding domain to theremainder of the delivery construct with one or more non-covalentinteractions of sufficient affinity. One of skill in the art can readilyrecognize linkers that interact with each other with sufficient affinityto be useful in the delivery constructs of the invention. For example,biotin can be attached to the receptor binding domain, and streptavidincan be attached to the remainder of the molecule. In certainembodiments, the linker can directly link the receptor binding domain tothe remainder of the molecule. In other embodiments, the linker itselfcomprises two or more molecules that associate in order to link thereceptor binding domain to the remainder of the molecule. Exemplarylinkers include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, substituted carbon linkers,unsaturated carbon linkers, aromatic carbon linkers, peptide linkers,etc.

In embodiments where a linker is used to connect the receptor bindingdomain to the remainder of the delivery construct, the linkers can beattached to the receptor binding domain and/or the remainder of thedelivery construct by any means or method known by one of skill in theart without limitation. For example, the linker can be attached to thereceptor binding domain and/or the remainder of the delivery constructwith an ether, ester, thioether, thioester, amide, imide, disulfide,peptide, or other suitable moiety. The skilled artisan can select theappropriate linker and method for attaching the linker based on thephysical and chemical properties of the chosen receptor binding domainand the linker. The linker can be attached to any suitable functionalgroup on the receptor binding domain or the remainder of the molecule.For example, the linker can be attached to sulfhydryl (—S), carboxylicacid (COOH) or free amine (—NH₂) groups, which are available forreaction with a suitable functional group on a linker. These groups canalso be used to connect the receptor binding domain directly connectedwith the remainder of the molecule in the absence of a linker.

Further, the receptor binding domain and/or the remainder of thedelivery construct can be derivatized in order to facilitate attachmentof a linker to these moieties. For example, such derivatization can beaccomplished by attaching suitable derivative such as those availablefrom Pierce Chemical Company, Rockford, Ill. Alternatively,derivatization may involve chemical treatment of the receptor bindingdomain and/or the remainder of the molecule. For example, glycolcleavage of the sugar moiety of a carbohydrate or glycoprotein receptorbinding domain with periodate generates free aldehyde groups. These freealdehyde groups may be reacted with free amine or hydrazine groups onthe remainder of the molecule in order to connect these portions of themolecule. See, e.g., U.S. Pat. No. 4,671,958. Further, the skilledartisan can generate free sulfhydryl groups on proteins to provide areactive moiety for making a disulfide, thioether, thioester, etc.linkage. See, e.g., U.S. Pat. No. 4,659,839.

Any of these methods for attaching a linker to a receptor binding domainand/or the remainder of a delivery construct can also be used to connecta receptor binding domain with the remainder of the delivery constructin the absence of a linker. In such embodiments, the receptor bindingdomain is coupled with the remainder of the construct using a methodsuitable for the particular receptor binding domain. Thus, any methodsuitable for connecting a protein, peptide, polypeptide, nucleic acid,carbohydrate, lipid, or small organic molecule to the remainder of thedelivery construct known to one of skill in the art, without limitation,can be used to connect the receptor binding domain to the remainder ofthe construct. In addition to the methods for attaching a linker to areceptor binding domain or the remainder of a delivery construct, asdescribed above, the receptor binding domain can be connected with theremainder of the construct as described, for example, in U.S. Pat. Nos.6,673,905; 6,585,973; 6,596,475; 5,856,090; 5,663,312; 5,391,723;6,171,614; 5,366,958; and 5,614,503.

In certain embodiments, the receptor binding domain can be a monoclonalantibody. In some of these embodiments, the chimeric immunogen isexpressed as a fusion protein that comprises an immunoglobulin heavychain from an immunoglobulin specific for a receptor on a cell to whichthe chimeric immunogen is intended to bind. The light chain of theimmunoglobulin then can be co-expressed with the chimeric immunogen,thereby forming a light chain-heavy chain dimer. In other embodiments,the antibody can be expressed and assembled separately from theremainder of the chimeric immunogen and chemically linked thereto.

5.2.2. Transcytosis Domain

The delivery constructs of the invention also comprise a transcytosisdomain. The transcytosis domain can be any transcytosis domain known byone of skill in the art to effect transcytosis of chimeric proteins thathave bound to a cell surface receptor present on the apical membrane ofan epithelial cell. In certain embodiments, the transcytosis domain is atranscytosis domain from PE, diptheria toxin, pertussis toxin, choleratoxin, heat-labile E. coli enterotoxin, shiga toxin, or shiga-liketoxin. See, for example, U.S. Pat. Nos. 5,965,406, and 6,022,950. Inpreferred embodiments, the transcytosis domain is domain II of PE.

The transcytosis domain need not, though it may, comprise the entireammo acid sequence of domain II of native PE, which spans residues253-364 of PE. For example, the transcytosis domain can comprise aportion of PE that spans residues 280-344 of domain II of PE. The aminoacids at positions 339 and 343 appear to be necessary for transcytosis.See Siegall et al., 1991, Biochemistry 30:7154-59. Further, conservativeor nonconservative substitutions can be made to the amino acid sequenceof the transcytosis domain, as long as transcytosis activity is notsubstantially eliminated. A representative assay that can routinely beused by one of skill in the art to determine whether a transcytosisdomain has transcytosis activity is described below.

Without intending to be limited to any particular theory or mechanism ofaction, the transcytosis domain is believed to permit the trafficking ofthe delivery construct through a polarized epithelial cell after theconstruct binds to a receptor present on the apical surface of thepolarized epithelial cell. Such trafficking through a polarizedepithelial cell is referred to herein as “transcytosis.” Thistrafficking permits the release of the delivery construct from thebasal-lateral membrane of the polarized epithelial cell.

5.2.3. Macromolecules for Delivery

The delivery constructs of the invention can also comprise amacromolecule. The macromolecule can be attached to the remainder of thedelivery construct by any method known by one of skill in the art,without limitation. In certain embodiments, the macromolecule isexpressed together with the remainder of the delivery construct as afusion protein. In such embodiments, the macromolecule can be insertedinto or attached to any portion of the delivery construct, so long asthe receptor binding domain, the transcytosis domain, and macromoleculeretain their activities. The macromolecule is connected with theremainder of the construct with a cleavable linker, or a combination ofcleavable linkers, as described below.

In native PE, the Ib loop (domain Ib) spans amino acids 365 to 399, andis structurally characterized by a disulfide bond between two cysteinesat positions 372 and 379. This portion of PE is not essential for anyknown activity of PE, including cell binding, transcytosis, ER retentionor ADP ribosylation activity. Accordingly, domain Ib can be deletedentirely, or modified to contain a macromolecule.

Thus, in certain embodiments, the macromolecule can be inserted intodomain Ib. If desirable, the macromolecule can be inserted into domainIb wherein the cysteines at positions 372 and 379 are not cross-linked,this can be accomplished by reducing the disulfide linkage between thecysteines, by deleting the cysteines entirely from the Ib domain, bymutating the cysteines to other residues, such as, for example, serine,or by other similar techniques. Alternatively, the macromolecule can beinserted into the Ib loop between the cysteines at positions 372 and379. In such embodiments, the disulfide linkage between the cysteinescan be used to constrain the macromolecule if desirable. In any event,in embodiments where the macromolecule is inserted into domain Ib of PE,or into any other portion of the delivery construct, the macromoleculeshould be flanked by cleavable linkers such that cleavage at thecleavable linkers liberates the macromolecule from the remainder of theconstruct.

In other embodiments, the macromolecule can be connected with theN-terminal or C-terminal end of a polypeptide portion of the deliveryconstruct. In such embodiments, the method of connection should bedesigned to avoid interference with other functions of the deliveryconstruct, such as receptor binding or transcytosis. In yet otherembodiments, the macromolecule can be connected with a side chain of anamino acid of the delivery construct. The macromolecule is connectedwith the remainder of the delivery construct with a cleavable linker, asdescribed below. In such embodiments, the macromolecule to be deliveredcan be connected with the remainder of the delivery construct with oneor more cleavable linkers such that cleavage at the cleavable linker(s)separates the macromolecule from the remainder of the deliveryconstruct. It should be noted that, in certain embodiments, themacromolecule of interest can also comprise a short (1-20 amino acids,preferably 1-10 amino acids, and more preferably 1-5 amino acids) leaderpeptide in addition to the macromolecule of interest that remainsattached to the macromolecule following cleavage of the cleavablelinker. Preferably, this leader peptide does not affect the activity orimmunogenicity of the macromolecule.

In embodiments where the macromolecule is expressed together withanother portion of the delivery construct as a fusion protein, themacromolecule can be can be inserted into the delivery construct by anymethod known to one of skill in the art without limitation. For example,amino acids corresponding to the macromolecule can be inserted directlyinto the delivery construct, with or without deletion of native aminoacid sequences. In certain embodiments, all or part of the Ib domain ofPE can be deleted and replaced with the macromolecule. In certainembodiments, the cysteine residues of the Ib loop are deleted so thatthe macromolecule remains unconstrained. In other embodiments, thecysteine residues of the Ib loop are linked with a disulfide bond andconstrain the macromolecule.

The macromolecule can be any macromolecule that is desired to beintroduced into a subject. Thus, the macromolecule can be a peptide, apolypeptide, a protein, a nucleic acid, a carbohydrate, a lipid, aglycoprotein, synthetic organic and inorganic compounds, or anycombination thereof. The macromolecule can also be a detectable compoundsuch as a radiopaque compound, including air and barium and magneticcompounds. In certain embodiments, the macromolecule can be eithersoluble or insoluble in water. In certain embodiments, the macromoleculecan be a macromolecule that can perform a desirable biological activitywhen introduced to the bloodstream of the subject. For example, themacromolecule can have receptor binding activity, enzymatic activity,messenger activity (i.e., act as a hormone, cytokine, neurotransmitter,or other signaling molecule), luminescent or other detectable activity,or regulatory activity, or any combination thereof. In, for example,diagnostic embodiments, the macromolecule can be conjugated to or canitself be a pharmaceutically acceptable gamma-emitting moiety, includingbut not limited to, indium and technetium, magnetic particles,radiopaque materials such as air or barium and fluorescent compounds.

In other embodiments, the macromolecule that is delivered can exert itseffects in biological compartments of the subject other than thesubject's blood. For example, in certain embodiments, the macromoleculecan exert its effects in the lymphatic system. In other embodiments, themacromolecule can exert its effects in an organ or tissue, such as, forexample, the subject's liver, heart, lungs, pancreas, kidney, brain,bone marrow, etc. In such embodiments, the macromolecule may or may notbe present in the blood, lymph, or other biological fluid at detectableconcentrations, yet may still accumulate at sufficient concentrations atits site of action to exert a biological effect.

Further, the macromolecule can be a protein that comprises more than onepolypeptide subunit. For example, the protein can be a dimer, trimer, orhigher order multimer. In certain embodiments, two or more subunits ofthe protein can be connected with a covalent bond, such as, for example,a disulfide bond. In other embodiments, the subunits of the protein canbe held together with non-covalent interactions. One of skill in the artcan routinely identify such proteins and determine whether the subunitsare properly associated using, for example, an immunoassay. Exemplaryproteins that comprise more than one polypeptide chain that can bedelivered with a delivery construct of the invention include, but arenot limited to, antibodies, insulin, IGF I, and the like.

Accordingly, in certain embodiments, the macromolecule is a peptide,polypeptide, or protein. In certain embodiments, the macromoleculecomprises a peptide or polypeptide that comprises about 5, about 8,about 10, about 12, about 15, about 17, about 20, about 25, about 30,about 40, about 50, or about 60, about 70, about 80, about 90, about100, about 200, about 400, about 600, about 800, or about 1000 aminoacids. In certain embodiments, the macromolecule is a protein thatcomprises 1, 2, 3, 4, 5, 6, 7, 8, or more polypeptides. In certainembodiments, the peptide, polypeptide, or protein is a molecule that iscommonly administered to subjects by injection. Exemplary peptides orpolypeptides include, but are not limited to, IGF-I, IGF-II, IGF-III,EGF, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-6, IL-8,IL-12, EPO, growth hormone, clotting factors such as factor VII,vasopressin, calcitonin parathyroid hormone, luteinizinghormone-releasing factor, tissue plasminogen activators,adrenocorticototropin, enkephalin, glucagon-like peptide 1,asparaginase, and the like. In a preferred embodiment, the macromoleculeis insulin. In certain preferred embodiments, the polypeptide is growthhormone. In even more preferred embodiments, the polypeptide is humangrowth hormone. In an equally preferred embodiment, the polypeptide isIFN-α, more preferably IFNα-2b. In an equally preferred embodiment, thepolypeptide is insulin or proinsulin. In other embodiments, thepolypeptide is green fluorescent protein. The sequences of all of thesemacromolecules are well known to those in the art, and attachment ofthese macromolecules to the delivery constructs is well within the skillof those in the art using standard techniques, as discussed below.

In certain embodiments, the macromolecule can be selected to not becleavable by an enzyme present at the basal-lateral membrane of anepithelial cell. For example, the assays described in the examples canbe used to routinely test whether such a cleaving enzyme can cleave themacromolecule to be delivered. If so, the macromolecule can be routinelyaltered to eliminate the offending amino acid sequence recognized by thecleaving enzyme. The altered macromolecule can then be tested to ensurethat it retains activity using methods routine in the art.

Other examples of macromolecules that can be delivered according to thepresent invention include, but are not limited to, antineoplasticcompounds, such as nitrosoureas, e.g., carmustine, lomustine, semustine,strepzotocin; methylhydrazines, e.g., procarbazine, dacarbazine; steroidhormones, e.g., glucocorticoids, estrogens, progestins, androgens,tetrahydrodesoxycaricosterone; immunoactive compounds such asimmunosuppressives, e.g., pyrimemamine, trimethopterin, pemcillamine,cyclosporine, azathioprine; and immunostimulants, e.g., levamisole,diethyl dithiocarbamate, enkephalins, endorphins; antimicrobialcompounds such as antibiotics, e.g., β-lactam, penicillin,cephalosporins, carbapenims and monobactams, β-lactamase inhibitors,aminoglycosides, macrolides, tetracyclins, spectinomycin; antimalarials,amebicides; antiprotazoals; antifungals, e.g., amphotericin β,antivirals, e.g., acyclovir, idoxuridine, ribavirin, trifluridine,vidarbine, gancyclovir; parasiticides; antihalmintics;radiopharmaceutics; gastrointestinal drugs; hematologic compounds;immunoglobulins; blood clotting proteins, e.g., antihemophilic factor,factor IX complex; anticoagulants, e.g., dicumarol, heparinNa;fibrolysin inhibitors, e.g., tranexamic acid; cardiovascular drugs;peripheral anti-adrenergic drugs; centrally acting antihypertensivedrugs, e.g., methyldopa, methyldopa HCl; antihypertensive directvasodilators, e.g., diazoxide, hydralazine HCl; drugs affectingrenin-angiotensin system; peripheral vasodilators, e.g., phentolamine;anti-anginal drugs; cardiac glycosides; inodilators, e.g., amrinone,milrinone, enoximone, fenoximone, imazodan, sulmazole; antidysrhythmics;calcium entry blockers; drugs affecting blood lipids, e.g., ranitidine,bosentan, rezulin; respiratory drugs; sypamomimetic drugs, e.g.,albuterol, bitolterol mesylate, dobutamine HCl, dopamine HCl, ephedrineSo, epinephrine, fenfluramine HCl, isoproterenol HCl, methoxamine HCl,norepinephrine bitartrate, phenylephrine HCl, ritodrine HCl;cholmomimetic drugs, e.g., acetylcholine Cl; anticholinesterases, e.g.,edrophonium Cl; cholinesterase reactivators; adrenergic blocking drugs,e.g., acebutolol HCl, atenolol, esmolol HCl, labetalol HCl, metoprolol,nadolol, phentolamine mesylate, propanolol HCl; antimuscarinic drugs,e.g., anisotropine methylbromide, atropine SO₄, clinidium Br,glycopyrrolate, ipratropium Br, scopolamine HBr; neuromuscular blockingdrugs; depolarizing drugs, e.g., atracurium besylate, hexafluorenium Br,metocurine iodide, succinylcholine Cl, tubocurarine Cl, vecuronium Br;centrally acting muscle relaxants, e.g., baclofen; neurotransmitters andneurotransmitter agents, e.g., acetylcholine, adenosine, adenosinetriphosphate; amino acid neurotransmitters, e.g., excitatory aminoacids, GABA, glycine; biogenic amine neurotransmitters, e.g., dopamine,epinephrine, histamine, norepinephrine, octopamine, serotonin, tyramine;neuropeptides, nitric oxide, K⁺ channel toxins; antiparkinson drugs,e.g., amaltidine HCl, benztropine mesylate, carbidopa; diuretic drugs,e.g., dichlorphenamide, methazolamide, bendroflumethiazide,polythiazide; antimigraine drugs, e.g, carboprost tromethamine mesylate,methysergide maleate.

Still other examples of macromolecules that can be delivered accordingto the present invention include, but are not limited to, hormones suchas pituitary hormones, e.g., chorionic gonadotropin, cosyntropin,menotropins, somatotropin, iorticotropin, protirelin, thyrotropin,vasopressin, lypressin; adrenal hormones, e.g., beclomethasonedipropionate, betamethasone, dexarnethasone, triamcinolone; pancreatichormones, e.g., glucagon, insulin; parathyroid hormone, e.g.,dihydrochysterol; thyroid hormones, e.g., calcitonin etidronatedisodium, levothyroxine Na, liothyronine Na, liotrix, thyroglobulin,teriparatide acetate; antithyroid drugs; estrogenic hormones; progestinsand antagonists; hormonal contraceptives; testicular hormones;gastrointestinal hormones, e.g., cholecystokinin, enteroglycan, galanin,gastric inhibitory polypeptide, epidermal growth factor-urogastrone,gastric inhibitory polypeptide, gastrin-releasing peptide, gastrins,pentagastrin, tetragastrin, motilin, peptide YY, secretin, vasoactiveintestinal peptide, sincalide.

Still other examples of macromolecules that can be delivered accordingto the present invention include, but are not limited to, enzymes suchas hyaluronidase, streptokinase, tissue plasminogen activator,urokinase, PGE-adenosine deaminase; intravenous anesthetics such asdroperidol, etomidate, fetanyl citrate/droperidol, hexobarbital,ketamine HCl, methohexital Na, thiamylal Na, thiopental Na;antiepileptics, e.g., carbamazepine, clonazepam, divalproex Na,ethosuximide, mephenyloin, paramethadione, phenyloin, primidone.

Still other examples of macromolecules that can be delivered accordingto the present invention include, but are not limited to, peptides andproteins such as ankyrins, arrestins, bacterial membrane proteins,clathrin, connexins, dystrophin, endothelin receptor, spectrin,selectin, cytokines; chemokines; growth factors, insulin, erythropoietin(EPO), tumor necrosis factor (TNF), neuropeptides, neuropeptide Y,neurotensin, transforming growth factor α, transforming growth factor β,interferon (IFN); hormones, growth inhibitors, e.g., genistein, steroidsetc; glycoproteins, e.g., ABC transporters, platelet glycoproteins,GPIb-IX complex, GPIIb-IIIa complex, vitronectin, thrombomodulin, CD4,CD55, CD58, CD59, CD44, lymphocye function-associated antigen,intercellular adhesion molecule, vascular cell adhesion molecule, Thy-1,antiporters, CA-15-3 antigen, fibronectins, laminin, myelin-associatedglycoprotein, GAP, GAP-43.

Yet other examples of macromolecules that can be delivered according tothe present invention include, but are not limited to, cytokines andcytokine receptors such as Interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-4receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor,IL-9 receptor, IL-10 receptor, IL-11 receptor, IL-12 receptor, IL-13receptor, IL-14 receptor, IL-15 receptor, IL-16 receptor, IL-17receptor, IL-18 receptor, lymphokine inhibitory factor, macrophagecolony stimulating factor, platelet derived growth factor, stem cellfactor, tumor growth factor β, tumor necrosis factor, lymphotoxin, Fas,granulocyte colony stimulating factor, granulocyte macrophage colonystimulating factor, interferon α, interferon β, and interferon γ.

Still other examples of macromolecules that can be delivered accordingto the present invention include, but are not limited to, growth factorsand protein hormones such as erythropoietin, angiogenin, hepatocytegrowth factor, fibroblast growth factor, keratinocyte growth factor,nerve growth factor, tumor growth factor α, thrombopoietin, thyroidstimulating factor, thyroid releasing hormone, neurotrophin, epidermalgrowth factor, VEGF, ciliary neurotrophic factor, LDL, somatomedin,insulin growth factor, insulin-like growth factor I and II; chemokinessuch as ENA-78, ELC, GRO-α, GRO-β, GRO-γ, HRG, LEF, IP-10, MCP-1, MCP-2,MCP-3, MCP-4, MIP-1α, MIP-1β, MG, MDC, NT-3, NT-4, SCF, LIF, leptin,RANTES, lymphotactin, eotaxin-1, eotaxin-2, TARC, TECK, WAP-1, WAP-2,GCP-1, GCP-2; α-chemokine receptors, e.g., CXCR1, CXCR2, CXCR3, CXCR4,CXCR5, CXCR6, CXCR7; and β-chemokine receptors, e.g., CCR1, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7.

Yet other examples of macromolecules that can be delivered according tothe present invention include, but are not limited to,chemotherapeutics, such as chemotherapy or anti-tumor agents which areeffective against various types of human cancers, including leukemia,lymphomas, carcinomas, sarcomas, myelomas etc., such as, for example,doxorubicin, mitomycin, cisplatin, daunorubicin, bleomycin, actinomycinD, and neocarzinostatin.

Still other examples of macromolecules that can be delivered accordingto the present invention include, but are not limited to, antibodiessuch as anti-cluster of differentiation antigen CD-1 through CD-166 andthe ligands or counter receptors for these molecules; anti-cytokineantibodies, e.g., anti-IL-1 through anti-IL-18 and the receptors forthese molecules; anti-immune receptor antibodies; antibodies against Tcell receptors, major histocompatibility complexes I and II, B cellreceptors, selectin killer inhibitory receptors, killer activatingreceptors, OX-40, MadCAM-1, Gly-CAM2, integrins, cadherens,sialoadherens, Fas, CTLA-4, Fc γ-receptors, Fc α-receptors, Fcε-receptors, Fc μ-receptors, and their ligands; anti-metalloproteinaseantibodies, e.g., antibodies specific for collagenase, MMP-1 throughMMP-8, TIMP-1, TIMP-2; anti-cell lysis/proinflammatory molecules, e.g.,perforin, complement components, prostanoids, nitron oxide,thromboxanes; and anti-adhesion molecules, e.g., carcioembryonicantigens, lamins, fibronectins.

Yet other examples of macromolecules that can be delivered according tothe present invention include, but are not limited to, antiviral agentssuch as reverse transcriptase inhibitors and nucleoside analogs, e.g.,ddI, ddC, 3TC, ddA, AZT; protease inhibitors, e.g., Invirase, ABT-538;and inhibitors of in RNA processing, e.g., ribavirin.

Further, specific examples of macromolecules that can be delivered withthe delivery constructs of the present invention Capoten, Monopril,Pravachol, Avapro, Plavix, Cefzil, Duricef/Ultracef, Azactam, Videx,Zerit, Maxipime, VePesid, Paraplatin, Platinol, Taxol, UFT, Buspar,Serzone, Stadol NS, Estrace, Glucophage (Bristol-Myers Squibb); Ceclor,Lorabid, Dynabac, Prozac, Darvon, Permax, Zyprexa, Humalog, Axid,Gemzar, Evista (Eli Lily); Vasotec/Vaseretic, Mevacor, Zocor,Prinivil/Prinizide, Plendil, Cozaar/Hyzaar, Pepcid, Prilosec, Primaxin,Noroxin, Recombivax HB, Varivax, Timoptic/XE, Trusopt, Proscar, Fosamax,Sinemet, Crixivan, Propecia, Vioxx, Singulair, Maxalt, Ivermectin (Merck& Co.); Diflucan, Unasyn, Sulperazon, Zithromax, Trovan, Procardia XL,Cardura, Norvasc, Dofetilide, Feldene, Zoloft, Zeldox, Glucotrol XL,Zyrtec, Eletriptan, Viagra, Droloxifene, Aricept, Lipitor (Pfizer);Vantin, Rescriptor, Vistide, Genotropin, Micronase/Glyn./Glyb., Fragmin,Total Medrol, Xanax/alprazolam, Sermion, Halcion/triazolam, Freedox,Dostinex, Edronax, Mirapex, Pharmorubicin, Adriamycin, Camptosar,Remisar, Depo-Provera, Caverject, Detrusitol, Estring, Healon, Xalatan,Rogaine (Pharmacia & Upjohn); Lopid, Accrupil, Dilantin, Cognex,Neurontin, Loestrin, Dilzem, Fempatch, Estrostep, Rezulin, Lipitor,Omnicef, FemHRT, Suramin, and Clinafloxacin (Warner Lambert).

Yet further examples of macromolecules which may be delivered by thedelivery constructs of the present invention may be found in: Goodmanand Gilman's The Pharmacological Basis of Therapeutics, 9th ed.McGraw-Hill 1996, incorporated herein by reference in its entirety.

In certain embodiments, the macromolecule can be inactive or in a lessactive form when administered, then be activated in the subject. Forexample, the macromolecule can be a peptide or polypeptide with a maskedactive site. The peptide or polypeptide can be activated by removing themasking moiety. Such removal can be accomplished by peptidases orproteases in the cases of peptide or polypeptide masking agents.Alternatively, the masking agent can be a chemical moiety that isremoved by an enzyme present in the subject. This strategy can be usedwhen it is desirable for the macromolecule to be active in limitedcircumstances. For example, it may be useful for a macromolecule to beactive only in the liver of the subject. In such cases, themacromolecule can be selected to have a masking moiety that can beremoved by an enzyme that is present in the liver, but not in otherorgans or tissues. Exemplary methods and compositions for making andusing such masked macromolecules can be found in U.S. Pat. Nos.6,080,575, 6,265,540, and 6,670,147.

In another example of such embodiments, the macromolecule can be apro-macromolecule that is activated by a biological activity, forexample by processing, present in the subject. For example, theexemplary macromolecule proinsulin can be delivered with a deliveryconstruct of the present invention. Following delivery of thepro-macromolecule, it can be activated in the subject by appropriateprocessing enzymes. While it is believed that proinsulin is processed byenzymes (the endoproteases PC2 and PC3) present in highest concentrationin secretory granules of pancreatic beta-cells, it is also believed thatsuch enzyme are present in sufficient concentration in othercompartments to permit activation of the pro-macromolecule into itsfully active form. Further, it should be noted that manypro-macromolecules, including, for example, proinsulin, also exhibitactivity similar to that of the fully active molecule. See, for example,Desbuquois et al., 2003, Endocrinology 12:5308-5321. Thus, even if notall of the pro-macromolecule is converted to the fully active form, thepro-molecule can in many cases still exert a desirable biologicalactivity in the subject.

5.2.4. Cleavable Linkers

In the delivery constructs of the invention, the macromolecule to bedelivered to the subject is connected with the remainder of the deliveryconstruct with one or more cleavable linkers. The number of cleavablelinkers present in the construct depends, at least in part, on thelocation of the macromolecule in relation to the remainder of thedelivery construct and the nature of the macromolecule. When themacromolecule is inserted into the delivery construct, the macromoleculecan be flanked by cleavable linkers, such that cleavage at both linkersseparates the macromolecule. The flanking cleavable linkers can be thesame or different from each other. When the macromolecule can beseparated from the remainder of the delivery construct with cleavage ata single linker, the delivery constructs can comprise a single cleavablelinker. Further, where the macromolecule is, e.g., a dimer or othermultimer, each subunit of the macromolecule can be separated from theremainder of the delivery construct and/or the other subunits of themacromolecule by cleavage at the cleavable linker.

The cleavable linkers are generally cleavable by a cleaving enzyme thatis present at or near the basal-lateral membrane of an epithelial cell.By selecting the cleavable linker to be cleaved by such enzymes, themacromolecule can be liberated from the remainder of the constructfollowing transcytosis across the mucous membrane and release from theepithelial cell into the cellular matrix on the basal-lateral side ofthe membrane. Further, cleaving enzymes could be used that are presentinside the epithelial cell, such that the cleavable linker is cleavedprior to release of the delivery construct from the basal-lateralmembrane, so long as the cleaving enzyme does not cleave the deliveryconstruct before the delivery construct enters the trafficking pathwayin the polarized epithelial cell that results in release of the deliveryconstruct and macromolecule from the basal-lateral membrane of the cell.

In certain embodiments, the cleaving enzyme is a peptidase. In otherembodiments, the cleaving enzyme is an RNAse. In yet other embodiments,the cleaving enzyme can cleave carbohydrates. Preferred peptidasesinclude, but are not limited to, Cathepsin GI, Chymotrypsin I, ElastaseI, Subtilisin AI, Subtilisin AII, Thrombin I, and Urokinase I. Table 1presents these enzymes together with an amino acid sequence that isrecognized and cleaved by the particular peptidase.

TABLE 1 Peptidases Present Near Basal-Lateral Mucous Membranes AminoAcid Sequence Peptidase Recognized and Cleaved Cathepsin GIAla-Ala-Pro-Phe (SEQ ID NO.: 4) Chymotrypsin I Gly-Gly-Phe (SEQ ID NO.:5) Elastase I Ala-Ala-Pro-Val (SEQ ID NO.: 6) Subtilisin AI Gly-Gly-Leu(SEQ ID NO.: 7) Subtilisin AII Ala-Ala-Leu (SEQ ID NO.: 8) Thrombin IPhe-Val-Arg (SEQ ID NO.: 9) Urokinase I Val-Gly-Arg (SEQ ID NO.: 10)

In certain embodiments, the delivery construct can comprise more thanone cleavable linker, wherein cleavage at either cleavable linker canseparate the macromolecule to be delivered from the delivery construct.In certain embodiments, the cleavable linker can be selected based onthe sequence, in the case of peptide, polypeptide, or proteinmacromolecules for delivery, to avoid the use of cleavable linkers thatcomprise sequences present in the macromolecule to be delivered. Forexample, if the macromolecule comprises AAL, the cleavable linker can beselected to be cleaved by an enzyme that does not recognize thissequence.

Further, the cleavable linker preferably exhibits a greater propensityfor cleavage than the remainder of the delivery construct. As oneskilled in the art is aware, many peptide and polypeptide sequences canbe cleaved by peptidases and proteases. In certain embodiments, thecleavable linker is selected to be preferentially cleaved relative toother amino acid sequences present in the delivery construct duringadministration of the delivery construct. In certain embodiments, thereceptor binding domain is substantially (e.g., about 99%, about 95%,about 90%, about 85%, about 80, or about 75%) intact following deliveryof the delivery construct to the bloodstream of the subject. In certainembodiments, the translocation domain is substantially (e.g., about 99%,about 95%, about 90%, about 85%, about 80, or about 75%) intactfollowing delivery of the delivery construct to the bloodstream of thesubject. In certain embodiments, the macromolecule is substantially(e.g., about 99%, about 95%, about 90%, about 85%, about 80, or about75%) intact following delivery of the delivery construct to thebloodstream of the subject. In certain embodiments, the cleavable linkeris substantially (e.g., about 99%, about 95%, about 90%, about 85%,about 80, or about 75%) cleaved following delivery of the deliveryconstruct to the bloodstream of the subject.

In other embodiments, the cleavable linker is cleaved by a cleavingenzyme found in the plasma of the subject. Any cleaving enzyme known byone of skill in the art to be present in the plasma of the subject canbe used to cleave the cleavable linker. Use of such enzymes to cleavethe cleavable linkers is less preferred than use of cleaving enzymesfound near the basal-lateral membrane of a polarized epithelial cellbecause it is believed that more efficient cleavage will occur in nearthe basal-lateral membrane. However, if the skilled artisan determinesthat cleavage mediated by a plasma enzyme is sufficiently efficient toallow cleavage of a sufficient fraction of the delivery constructs toavoid adverse effects, such plasma cleaving enzymes can be used tocleave the delivery constructs. Accordingly, in certain embodiments, thecleavable linker can be cleaved with an enzyme that is selected from thegroup consisting of caspase-1, caspase-3, proprotein convertase 1,proprotein convertase 2, proprotein convertase 4, proprotein convertase4 PACE 4, prolyl oligopeptidase, endothelin cleaving enzyme,dipeptidyl-peptidase IV, signal peptidase, neprilysin, renin, andesterase. See, e.g., U.S. Pat. No. 6,673,574. Table 2 presents theseenzymes together with an amino acid sequence(s) recognized by theparticular peptidase. The peptidase cleaves a peptide comprising thesesequences at the N-terminal side of the amino acid identified with anasterisk.

TABLE 2 Plasma Peptidases Amino Acid Sequence Peptidase Recognized andCleaved Caspase-1 Tyr-Val-Ala-Asp-Xaa* (SEQ ID NO.: 11) Caspase-3Asp-Xaa-Xaa-Asp-Xaa* (SEQ ID NO.: 12) Proprotein convertaseArg-(Xaa)_(n)-Arg-Xaa*; 1 n = 0, 2, 4 or 6 (SEQ ID NO.: 13) Proproteinconvertase Lys-(Xaa)_(n)-Arg-Xaa*; 2 n = 0, 2, 4, or 6 (SEQ ID NO.: 14)Proprotein convertase Glp-Arg-Thr-Lys-Arg-Xaa* 4 (SEQ ID NO.: 15)Proprotein convertase Arg-Val-Arg-Arg-Xaa* 4 PACE 4 (SEQ ID NO.: 16)Decanoyl-Arg-Val-Arg-Arg-Xaa* (SEQ ID NO.: 17) ProlyloligopeptidasePro-Xaa*-Trp-Val-Pro-Xaa Endothelin cleaving (SEQ ID NO.: 18) enzyme incombination with dipeptidyl- peptidase IV Signal peptidaseTrp-Val*-Ala-Xaa (SEQ ID NO.: 19) Neprilysin in com- Xaa-Phe*-Xaa-Xaabination with (SEQ ID NO.: 20) dipeptidyl-peptidase Xaa-Tyr*-Xaa-Xaa IV(SEQ ID NO.: 21) Xaa-Trp*-Xaa-Xaa (SEQ ID NO.: 22) Renin in combinationAsp-Arg-Tyr-Ile-Pro-Phe-His- with dipeptidyl- Leu*-Leu-(Val, Ala orPro)- peptidase IV Tyr-(Ser, Pro, or Ala) (SEQ ID NO.: 23)

Thus, in certain more preferred embodiments, the cleavable linker can beany cleavable linker known by one of skill in the art to be cleavable byan enzyme that is present at the basal-lateral membrane of an epithelialcell. In certain embodiments, the cleavable linker comprises a peptide.In other embodiments, the cleavable linker comprises a nucleic acid,such as RNA or DNA. In still other embodiments, the cleavable linkercomprises a carbohydrate, such as a disaccharide or a trisaccharide. Incertain embodiments, the cleavable linker is a peptide that comprises anamino acid sequence that is selected from the group consisting ofAla-Ala-Pro-Phe (SEQ ID NO.:4), Gly-Gly-Phe (SEQ ID NO.:5),Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu (SEQ ID NO.:7), Ala-Ala-Leu(SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9), Val-Gly-Arg (SEQ ID NO.:10).

Alternatively, in less preferred embodiments, the cleavable linker canbe any cleavable linker known by one of skill in the art to be cleavableby an enzyme that is present in the plasma of the subject to whom thedelivery construct is administered. In certain embodiments, thecleavable linker comprises a peptide. In other embodiments, thecleavable linker comprises a nucleic acid, such as RNA or DNA. In stillother embodiments, the cleavable linker comprises a carbohydrate, suchas a disaccharide or a trisaccharide. In certain embodiments, thecleavable linker is a peptide that comprises an amino acid sequence thatis selected from the group consisting of amino acid sequences presentedin Table 2.

In certain embodiments, the delivery construct comprises more than onecleavable linker. In certain embodiments, cleavage at any of thecleavable linkers will separate the macromolecule to be delivered fromthe remainder of the delivery construct. In certain embodiments, thedelivery construct comprises a cleavable linker cleavable by an enzymepresent at the basal-lateral side of a polarized epithelial membrane anda cleavable linkers cleavable by an enzyme that is present in the plasmaof the subject to whom the delivery construct is administered.

Further, Tables 4 and 5, below, present results of experiments testingthe ability of peptidases to cleave substrates when applied to thebasal-lateral or apical surface of a polarized epithelial membrane. Thesequences recognized by these enzymes are well-known in the art. Thus,in certain embodiments, the delivery construct comprises a cleavablelinker that is cleavable by an enzyme listed in Tables 4 and 5.Preferred peptidases exhibit higher activity on the basolateral side ofthe membrane. Particularly preferred peptidases exhibit much higher(e.g., 100%, 200%, or more increase in activity relative to the apicalside) on the basolateral side. Thus, in certain embodiments, thecleavable linker is cleavable by an enzyme that exhibits 50% higheractivity on the basal-lateral side of the membrane than on the apicalside of the membrane. In certain embodiments, the cleavable linker iscleavable by an enzyme that exhibits 100% higher activity on thebasal-lateral side of the membrane than on the apical side of themembrane. In certain embodiments, the cleavable linker is cleavable byan enzyme that exhibits 200% higher activity on the basal-lateral sideof the membrane than on the apical side of the membrane. In certainembodiments, the cleavable linker is cleavable by an enzyme thatexhibits 500% higher activity on the basal-lateral side of the membranethan on the apical side of the membrane. In certain embodiments, thecleavable linker is cleavable by an enzyme that exhibits 1,000% higheractivity on the basal-lateral side of the membrane than on the apicalside of the membrane. In certain embodiments, the cleavable linker iscleavable by an enzyme that exhibits 2,000% higher activity on thebasal-lateral side of the membrane than on the apical side of themembrane. In certain embodiments, the cleavable linker is cleavable byan enzyme that exhibits 3,000% higher activity on the basal-lateral sideof the membrane than on the apical side of the membrane. In certainembodiments, the cleavable linker is cleavable by an enzyme thatexhibits 5,000% higher activity on the basal-lateral side of themembrane than on the apical side of the membrane. In certainembodiments, the cleavable linker is cleavable by an enzyme thatexhibits 10,000% higher activity on the basal-lateral side of themembrane than on the apical side of the membrane.

Still further, the results in Tables 4 and 5 indicate that certainenzymes are present in higher concentration or exhibit greater activityin certain epithelial lineages as compared to other epithelial lineages.Thus, the experiments described below can be used to test whether theparticular epithelial cell lineage through which a macromolecule will bedelivered exhibits the desired cleavage activity. In certainembodiments, the cleavage activity is present in tracheal epithelialcells, but not intestinal epithelial cells. In other embodiments, thecleavage activity is present in intestinal epithelial cells out nottracheal epithelial cells. In certain embodiments, the cleavage activityis present in intestinal epithelial cells and tracheal epithelial cells.

In certain embodiments, the cleavable linker may be cleavable by anyenzyme that preferentially cleaves at the basolateral side of anepithelial membrane as compared to the apical side of the membrane.Example 6.4, below, describes an assay that can be used to assess theactivity of such enzymes, while Table 7, appended to the end of thisdocument, provides short names and accession numbers for every knownhuman protease or peptidase. Any cleavage sequence recognized by suchproteases or peptidases that preferentially cleaves a test substrate onthe basolateral side of an epithelial membrane, or in the plasma, ascompared to the apical side of such a membrane can also be used in themethods and compositions of the present invention. In such embodiments,one of skill in the art can readily determine the amino acid sequencerecognized by such peptidases or proteases according to standardprocedures known in the art or according to the known sequences,recognized by the proteases and peptidases.

The examples below provide methods for identifying cleaving enzymes thatare present at or near the basal-lateral membrane of a polarizedepithelial cell. The skilled artisan can routinely use such methods toidentify additional cleaving enzymes and the chemical structure(s)identified and cleaved by such cleaving enzymes. Delivery constructscomprising such cleavable linkers and methods of deliveringmacromolecules using delivery constructs comprising such cleavablelinkers are also within the scope of the present invention, whether ornot such cleaving enzymes are presented in Table 7.

In other embodiments, the cleavable linker can be a cleavable linkerthat is cleaved following a change in the environment of the deliveryconstruct. For example, the cleavable linker can be a cleavable linkerthat is pH sensitive and is cleaved by a change in pH that isexperienced when the delivery construct is released from thebasal-lateral membrane of a polarized epithelial cell. For instance, theintestinal lumen is strongly alkaline, while plasma is essentiallyneutral. Thus, a cleavable linker can be a moiety that is cleaved upon ashift from alkaline to neutral pH. The change in the environment of thedelivery construct that cleaves the cleavable linker can be anyenvironmental change that that is experienced when the deliveryconstruct is released from the basal-lateral membrane of a polarizedepithelial cell known by one of skill in the art, without limitation.

5.3. Methods for Delivering a Macromolecule

In another aspect, the invention provides methods for local or systemicdelivery of a macromolecule to a subject. These methods generallycomprise administering a delivery construct of the invention to a mucousmembrane of the subject to whom the macromolecule is delivered. Thedelivery construct is typically administered in the form of apharmaceutical composition, as described below.

Thus, in certain aspects, the invention provides a method for deliveringa macromolecule to a subject. The method comprises contacting an apicalsurface of a polarized epithelial cell of the subject with a deliveryconstruct. In certain embodiments, the delivery construct comprises areceptor binding domain, a transcytosis domain, a cleavable linker, andthe macromolecule to be delivered. The transcytosis domain cantranscytose the macromolecule to and through the basal-lateral membraneof said epithelial cell. The cleavable linker can be cleaved by ahenzyme that is present at a basal-lateral membrane of a polarizedepithelial cell of the subject or in the plasma of the subject. Cleavageat the cleavable linker separates the macromolecule from the remainderof the delivery construct, thereby delivering the macromolecule to thesubject.

In certain embodiments, the enzyme that is present at or near abasal-lateral membrane of a polarized epithelial cell is selected fromthe group consisting of Cathepsin GI, Chymotrypsin I, Elastase I,Subtilisin AI, Subtilisin AII, Thrombin I, and Urokinase I. In certainembodiments, the cleavable linker comprises an amino acid sequence thatis selected from the group consisting of Ala-Ala-Pro-Phe (SEQ ID NO.:4),Gly-Gly-Phe (SEQ ID NO.:5), Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu(SEQ ID NO.:7), Ala-Ala-Leu (SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9),Val-Gly-Arg (SEQ ID NO.:10).

In certain embodiments, the receptor binding domain is selected from thegroup consisting of receptor binding domains from Pseudomonas exotoxinA, cholera toxin, diptheria toxin, shiga toxin, or shiga-like toxin;monoclonal antibodies; polyclonal antibodies; single-chain antibodies;TGF α; EGF; IGF-I; IGF-II; IGF-III; IL-1; IL-2; IL-3; IL-6; MIP-1a;MIP-1b; MCAF; and IL-8. In certain embodiments, the receptor bindingdomain binds to a cell surface receptor selected from the groupconsisting of α2-macroglobulin receptor, EGFR, IGFR, transferrinreceptor, chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNFαreceptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavengerreceptor, and VEGF receptor.

In certain embodiments, the transcytosis domain is selected from thegroup consisting of transcytosis domains from Pseudomonas exotoxin A,diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. colienterotoxin, shiga toxin, and shiga-like toxin.

In certain embodiments, the macromolecule is selected from the groupconsisting of a peptide, a polypeptide, a protein, a nucleic acid, and alipid. In a preferred embodiment, the macromolecule is growth hormone.Even more preferably, the macromolecule is human growth hormone.

In certain embodiments, the invention provides a method for delivering amacromolecule to the bloodstream of a subject that results in at leastabout 30% bioavailability of the macromolecule, comprising administeringa delivery construct comprising the macromolecule to the subject,thereby delivering at least about 30% of the total macromoleculeadministered to the blood of the subject in a bioavailable form of themacromolecule. In certain embodiments, at least about 10% of the totalmacromolecule administered is bioavailable to the subject. In certainembodiments, at least about 15% of the total macromolecule administeredis bioavailable to the subject. In certain embodiments, at least about20% of the total macromolecule administered is bioavailable to thesubject. In certain embodiments, at least about 25% of the totalmacromolecule administered is bioavailable to the subject. In certainembodiments, at least about 35% of the total macromolecule administeredis bioavailable to the subject. In certain embodiments, at least about40% of the total macromolecule administered is bioavailable to thesubject. In certain embodiments, at least about 45% of the totalmacromolecule administered is bioavailable to the subject. In certainembodiments, at least about 50% of the total macromolecule administeredis bioavailable to the subject. In certain embodiments, at least about55% of the total macromolecule administered is bioavailable to thesubject. In certain embodiments, at least about 60% of the totalmacromolecule administered is bioavailable to the subject. In certainembodiments, at least about 65% of the total macromolecule administeredis bioavailable to the subject. In certain embodiments, at least about70% of the total macromolecule administered is bioavailable to thesubject. In certain embodiments, at least about 75% of the totalmacromolecule administered is bioavailable to the subject. In certainembodiments, at least about 80% of the total macromolecule administeredis bioavailable to the subject. In certain embodiments, at least about85% of the total macromolecule administered is bioavailable to thesubject. In certain embodiments, at least about 90% of the totalmacromolecule administered is bioavailable to the subject, in certainembodiments, at least about 95% of the total macromolecule administeredis bioavailable to the subject. In certain embodiments, the percentageof bioavailability of the macromolecule is determined by comparing theamount of macromolecule present in a subject's blood followingadministration of a delivery construct comprising the macromolecule tothe amount of macromolecule present in a subject's blood followingadministration of the macromolecule through another route ofadministration. In certain embodiments, the other route ofadministration is injection, e.g., subcutaneous injection, intravenousinjection, intra-arterial injection, etc. In other embodiments, thepercentage of bioavailability of the macromolecule is determined bycomparing the amount of macromolecule present in a subject's bloodfollowing administration of a delivery construct comprising themacromolecule to the total amount of macromolecule administered as partof the delivery construct.

In certain embodiments, peak plasma concentrations of the deliveredmacromolecule in the subject are achieved about 10 minutes afteradministration. In certain embodiments, peak plasma concentrations ofthe delivered macromolecule in the subject are achieved about 15 minutesafter administration. In certain embodiments, peak plasma concentrationsof the delivered macromolecule in the subject are achieved about 5minutes after administration. In certain embodiments, peak plasmaconcentrations of the delivered macromolecule in the subject areachieved about 20 minutes after administration. In certain embodiments,peak plasma concentrations of the delivered macromolecule in the subjectare achieved about 25 minutes after administration. In certainembodiments, peak plasma concentrations of the delivered macromoleculein the subject are achieved about 30 minutes after administration. Incertain embodiments, peak plasma concentrations of the deliveredmacromolecule in the subject are achieved about 35 minutes afteradministration. In certain embodiments, peak plasma concentrations ofthe delivered macromolecule in the subject are achieved about 40 minutesafter administration. In certain embodiments, peak plasma concentrationsof the delivered macromolecule in the subject are achieved about 45minutes after administration. In certain embodiments, peak plasmaconcentrations of the delivered macromolecule in the subject areachieved about 50 minutes after administration. In certain embodiments,peak plasma concentrations of the delivered macromolecule in the subjectare achieved about 55 minutes after administration. In certainembodiments, peak plasma concentrations of the delivered macromoleculein the subject are achieved about 60 minutes after administration. Incertain embodiments, peak plasma concentrations of the deliveredmacromolecule in the subject are achieved about 90 minutes alteradministration. In certain embodiments, peak plasma concentrations ofthe delivered macromolecule in the subject are achieved about 120minutes after administration.

In certain embodiments, the peak plasma concentration of the deliveredmacromolecule is between about 0.01 ng/ml plasma and about 10 μg/mlplasma. In certain embodiments, the peak plasma concentration of thedelivered macromolecule is between about 0.01 ng/ml plasma and about 1μg/ml plasma. In certain embodiments, the peak plasma concentration ofthe delivered macromolecule is between about 0.01 ng/ml plasma and about0.1 μg/ml plasma. In certain embodiments, the peak plasma concentrationof the delivered macromolecule is between about 0.01 ng/ml plasma andabout 10 ng/ml plasma. In certain embodiments, the peak plasmaconcentration of the delivered macromolecule is between about 1 ng/mlplasma and about 10 μg/ml plasma. In certain embodiments, the peakplasma concentration of the delivered macromolecule is between about 1ng/ml plasma and about 1 μg/ml plasma. In certain embodiments, the peakplasma concentration of the delivered macromolecule is between about 1ng/ml plasma and about 0.5 μg/ml plasma. In certain embodiments, thepeak plasma concentration of the delivered macromolecule is betweenabout 1 ng/ml plasma and about 0.1 μg/ml plasma. In certain embodiments,the peak plasma concentration of the delivered macromolecule is betweenabout 10 ng/ml plasma and about 1 μg/ml plasma. In certain embodiments,the peak plasma concentration of the delivered macromolecule is betweenabout 10 ng/ml plasma and about 0.5 μg/ml plasma.

In certain embodiments, the peak plasma concentration of the deliveredmacromolecule is at least about 10 ng/ml plasma. In certain embodiments,the peak plasma concentration of the delivered macromolecule is at leastabout 5 μg/ml plasma. In certain embodiments, the peak plasmaconcentration of the delivered macromolecule is at least about 1 μg/mlplasma. In certain embodiments, the peak plasma concentration of thedelivered macromolecule is at least about 500 ng/ml plasma. In certainembodiments, the peak plasma concentration of the deliveredmacromolecule is at least about 250 ng/ml plasma. In certainembodiments, the peak plasma concentration of the deliveredmacromolecule is at least about 100 ng/ml plasma. In certainembodiments, the peak plasma concentration of the deliveredmacromolecule is at least about 50 ng/ml plasma. In certain embodiments,the peak plasma concentration of the delivered macromolecule is at leastabout 10 ng/ml plasma. In certain embodiments, the peak plasmaconcentration of the delivered macromolecule is at least about 5 ng/mlplasma. In certain embodiments, the peak plasma concentration of thedelivered macromolecule is at least about 1 ng/ml plasma. In certainembodiments, the peak plasma concentration of the deliveredmacromolecule is at least about 0.1 ng/ml plasma.

Moreover, without intending to be bound to any particular theory ormechanism of action, it is believed that oral administration of adelivery construct can deliver a higher effective concentration of thedelivered macromolecule to the liver of the subject than is observed inthe subject's plasma. “Effective concentration,” in this context, refersto the concentration experienced by targets of the macromolecule and canbe determined by monitoring and/or quantifying downstream effects ofmacromolecule-target interactions. While still not bound to anyparticular theory, it is believed that oral administration of thedelivery construct results in absorption of the delivery constructthrough polarized epithelial cells of the digestive mucosa, e.g., theintestinal mucosa, followed by cleavage of the construct and release ofthe macromolecule at the basolateral side of the mucous membrane. As oneof skill in the art will recognize, the blood at the basolateralmembrane of such digestive mucosa is carried from this location to theliver via the portal venous system. Thus, when the macromolecule exertsa biological activity in the liver, such as, for example, activitiesmediated by growth hormone, insulin, IGF-I, etc. binding to theircognate receptors, the macromolecule is believed to exert an effect inexcess of what would be expected based on the plasma concentrationsobserved in the subject. Accordingly, in certain embodiments, theinvention provides a method of administering a macromolecule to asubject that comprises orally administering a delivery constructcomprising the macromolecule to the subject, wherein the macromoleculeis delivered to the subject's liver at a higher effective concentrationthan observed in the subject's plasma.

In certain embodiments, the epithelial cell is selected from the groupconsisting of nasal epithelial cells, oral epithelial cells, intestinalepithelial cells, rectal epithelial cells, vaginal epithelial cells, andpulmonary epithelial cells.

In certain embodiments, the subject is a mammal. In further embodiments,the subject is a rodent, a lagomorph, or a primate. In yet furtherembodiments, the rodent is a mouse or rat. In other embodiments, thelagomorph is a rabbit. In still other embodiments, the primate is ahuman, monkey, or ape. In a preferred embodiment, the subject is ahuman.

In another aspect, the invention provides a method for delivering amacromolecule to the bloodstream of a subject that induces a lower titerof antibodies against the macromolecule than other routes ofadministration. Without intending to be bound by any particular theoryor mechanism of action, it is believed that entry of the macromoleculethrough a mucous membrane, e.g., through the intestinal mucosa, causesthe immune system to tolerate the macromolecule better than if themacromolecule were, for example, injected. Thus, a lower titer ofantibodies against the macromolecule can be produced in the subject bydelivering the macromolecule with a delivery construct of the inventionthrough the mucosa rather than injecting the macromolecule, for example,subcutaneously, intravenously, intra-arterially, intraperitoneally, orotherwise. Generally, the time at which the lower titer of antibodiesdetected for the alternate routes of administration is detected shouldbe roughly comparable; for example, the titer of antibodies can bedetermined at about 1 week, at about 2 weeks, at about 3 weeks, at about4 weeks, at about 2 months, or at about 6 months followingadministration of the macromolecule with the delivery construct or byinjection.

Accordingly, in certain embodiments, the invention provides a method fordelivering a macromolecule to the bloodstream a subject that comprisescontacting a delivery construct of the invention that comprises themacromolecule to be delivered to an apical surface of a polarizedepithelial cell of the subject, such that the macromolecule isadministered to the bloodstream of the subject, wherein a lower titer ofantibodies specific for the macromolecule is induced in the serum of thesubject than is induced by subcutaneously administering themacromolecule separately from the remainder of the delivery construct toa subject.

In certain embodiments, the macromolecule is selected from the groupconsisting of a peptide, a polypeptide, a protein, a nucleic acid, and alipid. In certain embodiments, the macromolecule is selected from thegroup consisting of polypeptide hormones, cytokines, chemokines, growthfactors, and clotting factors. In certain embodiments, the macromoleculeis selected from the group consisting of IGF-I, IGF-II, IGF-III, EGF,IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-6, IL-8, IL-12,EPO, growth hormone, factor VII, vasopressin, calcitonin, parathyroidhormone, luteinizing hormone-releasing factor, tissue plasminogenactivators, adrenocorticototropin, enkephalin, and glucagon-likepeptide 1. In certain embodiments, the macromolecule is human growthhormone. In certain embodiments, the macromolecule is human insulin. Incertain embodiments, the subject is a mouse, rat, dog, goat, or human.

In certain embodiments, the titer of antibodies specific for themacromolecule induced in the serum of the subject by the macromoleculedelivered by the delivery construct is less than about 95% of the titerof antibodies induced by subcutaneously administering the macromoleculeseparately from the remainder of the delivery construct. In certainembodiments, the titer of antibodies specific for the macromoleculeinduced in the serum of the subject by the macromolecule delivered bythe delivery construct is less than about 90% of the titer of antibodiesinduced by subcutaneously administering the macromolecule separatelyfrom the remainder of the delivery construct. In certain embodiments,the titer of antibodies specific for the macromolecule induced in theserum of the subject by the macromolecule delivered by the deliveryconstruct is less than about 85% of the titer of antibodies induced bysubcutaneously administering the macromolecule separately from theremainder of the delivery construct. In certain embodiments, the titerof antibodies specific for the macromolecule induced in the serum of thesubject by the macromolecule delivered by the delivery construct is lessthan about 80% of the titer of antibodies induced by subcutaneouslyadministering the macromolecule separately from the remainder of thedelivery construct. In certain embodiments, the titer of antibodiesspecific for the macromolecule induced in the serum of the subject bythe macromolecule delivered by the delivery construct is less than about75% of the titer of antibodies induced by subcutaneously administeringthe macromolecule separately from the remainder of the deliveryconstruct.

In certain embodiments, the titer of antibodies specific for themacromolecule induced in the serum of the subject by the macromoleculedelivered by the delivery construct is less than about 70% of the titerof antibodies induced by subcutaneously administering the macromoleculeseparately from the remainder of the delivery construct. In certainembodiments, the titer of antibodies specific for the macromoleculeinduced in the serum of the subject by the macromolecule delivered bythe delivery construct is less than about 65% of the titer of antibodiesinduced by subcutaneously administering the macromolecule separatelyfrom the remainder of the delivery construct. In certain embodiments,the titer of antibodies specific for the macromolecule induced in theserum of the subject by the macromolecule delivered by the deliveryconstruct is less than about 60% of the titer of antibodies induced bysubcutaneously administering the macromolecule separately from theremainder of the delivery construct. In certain embodiments, the titerof antibodies specific for the macromolecule induced in the serum of thesubject by the macromolecule delivered by the delivery construct is lessthan about 55% of the titer of antibodies induced by subcutaneouslyadministering the macromolecule separately from the remainder of thedelivery construct. In certain embodiments, the titer of antibodiesspecific for the macromolecule induced in the serum of the subject bythe macromolecule delivered by the delivery construct is less than about55% of the titer of antibodies induced by subcutaneously administeringthe macromolecule separately from the remainder of the deliveryconstruct.

In certain embodiments, the titer of antibodies specific for themacromolecule induced in the serum of the subject by the macromoleculedelivered by the delivery construct is less than about 50% of the titerof antibodies induced by subcutaneously administering the macromoleculeseparately from the remainder of the delivery construct. In certainembodiments, the titer of antibodies specific for the macromoleculeinduced in the serum of the subject by the macromolecule delivered bythe delivery construct is less than about 45% of the titer of antibodiesinduced by subcutaneously administering the macromolecule separatelyfrom the remainder of the delivery construct. In certain embodiments,the titer of antibodies specific for the macromolecule induced in theserum of the subject by the macromolecule delivered by the deliveryconstruct is less than about 40% of the titer of antibodies induced bysubcutaneously administering the macromolecule separately from theremainder of the delivery construct. In certain embodiments, the titerof antibodies specific for the macromolecule induced in the serum of thesubject by the macromolecule delivered by the delivery construct is lessthan about 35% of the titer of antibodies induced by subcutaneouslyadministering the macromolecule separately from the remainder of thedelivery construct. In certain embodiments, the titer of antibodiesspecific for the macromolecule induced in the serum of the subject bythe macromolecule delivered by the delivery construct is less than about30% of the titer of antibodies induced by subcutaneously administeringthe macromolecule separately from the remainder of the deliveryconstruct.

In certain embodiments, the titer of antibodies specific for themacromolecule induced in the serum of the subject by the macromoleculedelivered by the delivery construct is less than about 25% of the titerof antibodies induced by subcutaneously administering the macromoleculeseparately from the remainder of the delivery construct. In certainembodiments, the titer of antibodies specific for the macromoleculeinduced in the serum of the subject by the macromolecule delivered bythe delivery construct is less than 20% of the titer of antibodiesinduced by subcutaneously administering the macromolecule separatelyfrom the remainder of the delivery construct. In certain embodiments,the titer of antibodies specific for the macromolecule induced in theserum of the subject by the macromolecule delivered by the deliveryconstruct is less than about 15% of the titer of antibodies induced bysubcutaneously administering the macromolecule separately from theremainder of the delivery construct. In certain embodiments, the titerof antibodies specific for the macromolecule induced in the serum of thesubject by the macromolecule delivered by the delivery construct is lessthan about 10% of the titer of antibodies induced by subcutaneouslyadministering the macromolecule separately from the remainder of thedelivery construct. In certain embodiments, the titer of antibodiesspecific for the macromolecule induced in the serum of the subject bythe macromolecule delivered by the delivery construct is less than about5% of the titer of antibodies induced by subcutaneously administeringthe macromolecule separately from the remainder of the deliveryconstruct. In certain embodiments, the titer of antibodies specific forthe macromolecule induced in the serum of the subject by themacromolecule delivered by the delivery construct is less than about 1%of the titer of antibodies induced by subcutaneously administering themacromolecule separately from the remainder of the delivery construct.

5.3.1. Methods of Administration

The delivery constructs of the invention can be administered to asubject by any method known to one of skill in the art. In certainembodiments, the delivery constructs are contacted to a mucosal membraneof the subject. For example, the mucosal membrane can be present in theeye, nose, mouth, trachea, lungs, esophagus, stomach, small intestine,large intestine, rectum, anus, sweat glands, vulva, vagina, or penis ofthe subject. Preferably, the mucosal membrane is a mucosal membranepresent in the digestive tract of the subject, such as a mucosalmembrane in the mouth, esophagus, stomach, small intestine, largeintestine, or rectum of the subject.

In such embodiments, the delivery constructs are preferably administeredto the subject orally. Thus, the delivery construct can be formulated toprotect the delivery construct from degradation in the acid environmentof the stomach, if necessary. For example, many embodiments of thedelivery constructs of the invention comprise polypeptide domains withdefined activities. Unless such delivery constructs are protected fromacid and/or enzymatic hydrolysis in the stomach, the constructs willgenerally be digested before delivery of substantial amounts of themacromolecule to be delivered. Accordingly, composition formulationsthat protect the delivery construct from degradation can be used inadministration of these delivery constructs.

5.3.2. Dosage

Generally, a pharmaceutically effective amount of the delivery constructof the invention is administered to a subject. The skilled artisan canreadily determine if the dosage of the delivery construct is sufficientto deliver an effective amount of the macromolecule, as described below.In certain embodiments, between about 1 μg and about 1 g of deliveryconstruct is administered. In other embodiments, between about 10 μg andabout 500 mg of delivery construct is administered. In still otherembodiments, between about 10 μg and about 100 mg of delivery constructis administered. In yet other embodiments, between about 10 μg and about1000 μg of delivery construct is administered. In still otherembodiments, between about 10 μg and about 250 μg of delivery constructis administered. In yet other embodiments, between about 10 μg and about100 μg of delivery construct is administered. Preferably, between about10 μg and about 50 μg of delivery construct is administered.

The volume of a composition comprising the delivery construct that isadministered will generally depend on the concentration of deliveryconstruct and the formulation of the composition. In certainembodiments, a unit dose of the delivery construct composition isbetween about 0.05 ml and about 1 ml, preferably about 0.5 ml. Thedelivery construct compositions can be prepared in dosage formscontaining between 1 and 50 doses (e.g., 0.5 ml to 25 ml), more usuallybetween 1 and 10 doses (e.g., 0.5 ml to 5 ml)

The delivery construct compositions of the invention can be administeredin one dose or in multiple doses. A dose can be followed by one or moredoses spaced by about 1 to about 6 hours, by about 6 to about 12 hours,by about 12 to about 24 hours, by about 1 day to about 3 days, by about1 day to about 1 week, by about 1 week to about 2 weeks, by about 2weeks to about 1 month, by about 4 to about 8 weeks, by about 1 to about3 months, or by about 1 to about 6 months.

The macromolecules to be delivered are generally macromolecules forwhich a large amount of knowledge regarding dosage, frequency ofadministration, and methods for assessing effective concentrations insubjects has accumulated. Such knowledge can be used to assessefficiency of delivery, effective concentration of the macromolecule inthe subject, and frequency of administration. Thus, the knowledge ofthose skilled in the art can be used to determine whether, for example,the amount of macromolecule delivered to the subject is an effectiveamount, the dosage should be increased or decreased, the subject shouldbe administered the delivery construct more or less frequently, and thelike.

5.3.3. Determining Amounts of Macromolecule Delivered

The methods of the invention can be used to deliver, either locally orsystemically, a pharmaceutically effective amount of a macromolecule toa subject. The skilled artisan can determine whether the methods resultin delivery of such a pharmaceutically effective amount of themacromolecule. The exact methods will depend on the macromolecule thatis delivered, but generally will rely on either determining theconcentration of the macromolecule in the blood of the subject or in thebiological compartment of the subject where the macromolecule exerts itseffects. Alternatively or additionally, the effects of the macromoleculeon the subject can be monitored.

For example, in certain embodiments of the present invention, themacromolecule that is delivered is insulin, e.g., human insulin. In suchembodiments, the skilled artisan can determine whether apharmaceutically effective amount of human insulin had been delivered tothe subject by, for example, taking a plasma sample from the subject anddetermining the concentration of human insulin therein. One exemplarymethod for determining the concentration of human insulin is byperforming an ELISA assay, but any other suitable assay known to theskilled artisan can be used.

Alternatively, one of skill in the art can determine if an effectiveamount of human insulin had been delivered to the subject by monitoringthe blood sugar concentrations of the subject. As is well-known in theart, human insulin, among other activities, acts on hepatocytes topromote glycogen formation, thereby reducing plasma glucoseconcentrations. Accordingly, the subject's plasma glucose concentrationcan be monitored to determine whether an effective amount of insulin hadbeen delivered.

Any effect of a macromolecule that is administered that is known by oneof skill in the art, without limitation, can be assessed in determiningwhether an effective amount of the macromolecule has been administered.Exemplary effects include, but are not limited to, receptor binding,receptor activation, downstream effects of receptor binding, downstreameffects of receptor activation, coordination of compounds, effectiveblood clotting, bone growth, wound healing, cellular proliferation, etc.The exact effect that is assessed will depend on the macromolecule thatis delivered.

5.4. Polynucleotides Encoding Delivery Constructs

In another aspect, the invention provides polynucleotides comprising anucleotide sequence encoding the delivery constructs. Thesepolynucleotides are useful, for example, for making the deliveryconstructs. In yet another aspect, the invention provides an expressionsystem that comprises a recombinant polynucleotide sequence encoding areceptor binding domain, a transcytosis domain, and a polylinkerinsertion site for a polynucleotide sequence encoding a macromolecule.The polylinker insertion site can be anywhere in the polynucleotidesequence so long as the polylinker insertion does not disrupt thereceptor binding domain or the transcytosis domain. The polylinkerinsertion site should be oriented near a polynucleotide sequence thatencodes a cleavable linker so that cleavage at the cleavable linkerseparates a macromolecule encoded by a nucleic acid inserted into thepolylinker insertion site from the remainder of the encoded deliveryconstruct. Thus, in embodiments where the polylinker insertion site isat an end of the encoded construct, the polynucleotide comprises onenucleotide sequence encoding a cleavable linker between the polylinkerinsertion site and the remainder of the polynucleotide. In embodimentswhere the polylinker insertion site is not at the end of the encodedconstruct, the polylinker insertion site can be flanked by nucleotidesequences that each encode a cleavable linker.

In certain embodiments, the recombinant polynucleotides are based onpolynucleotides encoding PE, or portions or derivatives thereof. Inother embodiments, the recombinant polynucleotides are based onpolynucleotides that hybridize to a polynucleotide that encodes PE understringent hybridization conditions. A nucleotide sequence encoding PE ispresented as SEQ ID NO.:3. This sequence can be used to prepare PCRprimers for isolating a nucleic acid that encodes any portion of thissequence that is desired. For example, PCR can be used to isolate anucleic acid that encodes one or more of the functional domains of PE. Anucleic acid so isolated can then be joined to nucleic acids encodingother functional domains of the delivery constructs using standardrecombinant techniques.

Other in vitro methods that can be used to prepare a polynucleotideencoding PE, PE domains, or any other functional domain useful in thedelivery constructs of the invention include, but are not limited to,reverse transcription, the polymerase chain reaction (PCR), the ligasechain reaction (LCR), the transcription-based amplification system(TAS), the self-sustained sequence replication system (3SR) and the QPreplicase amplification system (QB). Any such technique known by one ofskill in the art to be useful in construction of recombinant nucleicacids can be used. For example, a polynucleotide encoding the protein ora portion thereof can be isolated by polymerase chain reaction of cDNAusing primers based on the DNA sequence of PE or a nucleotide encoding areceptor binding domain.

Guidance for using these cloning and in vitro amplificationmethodologies are described in, for example, U.S. Pat. No. 4,683,195;Mullis et al., 1987, Cold Spring Harbor Symp. Quant. Biol. 51:263; andErlich, ed., 1989, PCR Technology, Stockton Press, NY. Polynucleotidesencoding a delivery construct or a portion thereof also can be isolatedby screening genomic or cDNA libraries with probes selected from thesequences of the desired polynucleotide under stringent, moderatelystringent, or highly stringent hybridization conditions.

Construction of nucleic acids encoding the delivery constructs of theinvention can be facilitated by introducing an insertion site for anucleic acid encoding the macromolecule into the construct. In certainembodiments, an insertion site for the macromolecule can be introducedbetween the nucleotides encoding the cysteine residues of domain Ib. Inother embodiments, the insertion site can be introduced anywhere in thenucleic acid encoding the construct so long as the insertion does notdisrupt the functional domains encoded thereby. In certain embodiments,the insertion site can be in the ER retention domain.

In more specific embodiments, a nucleotide sequence encoding a portionof the Ib domain between the cysteine-encoding residues can be removedand replaced with a nucleotide sequence that includes a cloning sitecleaved by a restriction enzyme. For example, the cloning site can berecognized and cleaved by PstI. In such examples, a polynucleotideencoding macromolecule that is flanked by PstI sequences can be insertedinto the vector.

Further, the polynucleotides can also encode a secretory sequence at theamino terminus of the encoded delivery construct. Such constructs areuseful for producing the delivery constructs in mammalian cells as theysimplify isolation of the immunogen.

Furthermore, the polynucleotides of the invention also encompassderivative versions of polynucleotides encoding a delivery construct.Such derivatives can be made by any method known by one of skill in theart without limitation. For example, derivatives can be made bysite-specific mutagenesis, including substitution, insertion, ordeletion of one, two, three, five, ten or more nucleotides, ofpolynucleotides encoding the delivery construct. Alternatively,derivatives can be made by random mutagenesis. One method for randomlymutagenizing a nucleic acid comprises amplifying the nucleic acid in aPCR reaction in the presence of 0.1 mM MnCl₂ and unbalanced nucleotideconcentrations. These conditions increase the misincorporation rate ofthe polymerase used in the PCR reaction and result in random mutagenesisof the amplified nucleic acid.

Several site-specific mutations and deletions in chimeric moleculesderived from PE have been made and characterized. For example, deletionof nucleotides encoding amino acids 1-252 of PE yields a constructreferred to as “PE40.” Deleting nucleotides encoding amino acids 1-279of PE yields a construct referred to as “PE37.” See U.S. Pat. No.5,602,095. In both of these constructs, the receptor binding domain ofPE, i.e., domain Ia, has been deleted. Nucleic acids encoding a receptorbinding domain can be ligated to these constructs to produce deliveryconstructs that are targeted to the cell surface receptor recognized bythe receptor binding domain. Of course, these recombinantpolynucleotides are particularly useful for expressing deliveryconstructs that have a receptor binding domain that is not domain Ia ofPE. The recombinant polynucleotides can optionally encode anamino-terminal methionine to assist in expression of the construct. Incertain embodiments, the receptor binding domain can be ligated to the5′ end of the polynucleotide encoding the transcytosis domain.

Other nucleic acids encoding mutant forms of PE that can be used as asource of nucleic acids for constructing the delivery constructs of theinvention include, but are not limited to, PEΔ553 and those described inU.S. Pat. Nos. 5,602,095; 5,512,658 and 5,458,878, and in Vasil et al.,1986, Infect. Immunol 52:538-48.

Accordingly, in certain embodiments, the invention provides apolynucleotide that encodes a delivery construct. The delivery constructcomprises a receptor binding domain, a transcytosis domain, amacromolecule to be delivered to a subject, and a cleavable linker.Cleavage at the cleavable linker can separate the macromolecule from theremainder of the construct. The cleavable linker can be cleaved by anenzyme that is present at a basal-lateral membrane of a polarizedepithelial cell of the subject or in the plasma of the subject.

In certain embodiments, the polynucleotide hybridizes under stringenthybridization conditions to any polynucleotide of this invention. Infurther embodiments, the polynucleotide hybridizes under stringentconditions to a nucleic acid that encodes any delivery construct of theinvention.

In certain embodiments, the polynucleotide encodes a delivery constructthat further comprises a second cleavable linker. In certainembodiments, the first and/or second cleavable linker comprises an aminoacid sequence that is selected from the group consisting ofAla-Ala-Pro-Phe (SEQ ID NO.:4), Gly-Gly-Phe (SEQ ID NO.:5),Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu (SEQ ID NO.:7), Ala-Ala-Leu(SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9), Val-Gly-Arg (SEQ ID NO.:10). In certain embodiments, the first and/or second cleavable linkerencoded by the polynucleotide is cleavable by an enzyme that is selectedfrom the group consisting of Cathepsin GI, Chymotrypsin I, Elastase I,Subtilisin AI, Subtilisin AII, Thrombin I, and Urokinase I.

In certain embodiments, the receptor binding domain encoded by thepolynucleotide is selected from the group consisting of receptor bindingdomains from Pseudomonas exotoxin A, cholera toxin, diptheria toxin,shiga toxin, or shiga-like toxin; monoclonal antibodies; polyclonalantibodies; single-chain antibodies; TGF α; EGF; IGF-I; IGF-II; IGF-III;IL-1; IL-2; IL-3; IL-6; MIP-1a; MIP-1b; MCAF; and IL-8. In certainembodiments, the receptor binding domain encoded by the polynucleotidebinds to a cell-surface receptor that is selected from the groupconsisting of α2-macroglobulin receptor, EGFR, IGFR, transferrinreceptor, chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNFαreceptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavengerreceptor, and VEGF receptor. In further embodiments, the receptorbinding domain encoded by the polynucleotide is Domain Ia of Pseudomonasexotoxin A. In yet further embodiments, the receptor binding domainencoded by the polynucleotide has an amino acid sequence that is SEQ IDNO.: 1.

In certain embodiments, the transcytosis domain encoded by thepolynucleotide is selected from the group consisting of transcytosisdomains from Pseudomonas exotoxin A, diptheria toxin, pertussis toxin,cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, andshiga-like toxin. In further embodiments, the transcytosis domain isPseudomonas exotoxin A transcytosis domain. In still furtherembodiments, the Pseudomonas exotoxin A transcytosis domain has an aminoacid sequence that is SEQ ID NO.:2.

In certain embodiments, the macromolecule encoded by the polynucleotideis selected from the group of a peptide, a polypeptide, and a protein.In certain embodiments, the polypeptide is selected from the groupconsisting of polypeptide hormones, cytokines, chemokines, growthfactors, and clotting factors. In further embodiments, the polypeptideis selected from the group consisting of IGF-I, IGF-II, IGF-III, EGF,IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-6, IL-8, IL-12,EPO, growth hormone, factor VII, vasopressin, calcitonin, parathyroidhormone, luteinizing hormone-releasing factor, tissue plasminogenactivators, adrenocorticototropin, enkephalin, and glucagon-likepeptide 1. In still further embodiments, the polypeptide is human growthhormone. In other embodiments, the protein is human insulin.

In other embodiments, the invention provides a polynucleotide thatencodes a delivery construct that comprises a nucleic acid sequenceencoding a receptor binding domain, a nucleic acid sequence encoding atranscytosis domain, a nucleic acid sequence encoding a cleavablelinker, and a nucleic acid sequence comprising a polylinker insertionsite. The polylinker insertion site can be oriented relative to thenucleic acid sequence encoding a cleavable linker to allow to cleavageof the cleavable linker to separate a macromolecule that is encoded by anucleic acid inserted into the polylinker insertion site from theremainder of said delivery construct. The cleavable linker can becleavable by an enzyme that is present at a basal-lateral membrane of apolarized epithelial cell of said subject or in the plasma of saidsubject.

5.5. Expression Vectors

In still another aspect, the invention provides expression vectors forexpressing the delivery constructs. Generally, expression vectors arerecombinant polynucleotide molecules comprising expression controlsequences operatively linked to a nucleotide sequence encoding apolypeptide. Expression vectors can readily be adapted for function inprokaryotes or eukaryotes by inclusion of appropriate promoters,replication sequences, selectable markers, etc. to result in stabletranscription and translation or mRNA. Techniques for construction ofexpression vectors and expression of genes in cells comprising theexpression vectors are well known in the art. See, e.g., Sambrook etal., 2001, Molecular Cloning—A Laboratory Manual, 3^(rd) edition, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., and Ausubel et al.,eds., Current Edition, Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, NY.

Useful promoters for use in expression vectors include, but are notlimited to, a metallothionein promoter, a constitutive adenovirus majorlate promoter, a dexamethasone-inducible MMTV promoter, a SV40 promoter,a MRP pol III promoter, a constitutive MPSV promoter, atetracycline-inducible CMV promoter (such as the human immediate-earlyCMV promoter), and a constitutive CMV promoter.

The expression vectors should contain expression and replication signalscompatible with the cell in which the delivery constructs are expressed.Expression vectors useful for expressing delivery constructs includeviral vectors such as retroviruses, adenoviruses and adenoassociatedviruses, plasmid vectors, cosmids, and the like. Viral and plasmidvectors are preferred for transfecting the expression vectors intomammalian cells. For example, the expression vector pcDNA1 (Invitrogen,San Diego, Calif.), in which the expression control sequence comprisesthe CMV promoter, provides good rates of transfection and expressioninto such cells.

The expression vectors can be introduced into the cell for expression ofthe delivery constructs by any method known to one of skill in the artwithout limitation. Such methods include, but are not limited to, e.g.,direct uptake of the molecule by a cell from solution; facilitateduptake through lipofection using, e.g., liposomes or immunoliposomes;particle-mediated transfection; etc. See, e.g., U.S. Pat. No. 5,272,065;Goeddel et al., eds, 1990, Methods in Enzymology, vol. 185, AcademicPress, Inc., CA; Krieger, 1990, Gene Transfer and Expression—ALaboratory Manual, Stockton Press, NY; Sambrook et al., 1989, MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, NY; andAusubel et al., eds., Current Edition, Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley Interscience, NY.

The expression vectors can also contain a purification moiety thatsimplifies isolation of the delivery construct. For example, apolyhistidine moiety of, e.g., six histidine residues, can beincorporated at the amino terminal end of the protein. The polyhistidinemoiety allows convenient isolation of the protein in a single step bynickel-chelate chromatography. In certain embodiments, the purificationmoiety can be cleaved from the remainder of the delivery constructfollowing purification. In other embodiments, the moiety does notinterfere with the function of the functional domains of the deliveryconstruct and thus need not be cleaved.

5.6. Cell for Expressing a Delivery Construct

In yet another aspect, the invention provides a cell comprising anexpression vector for expression of the delivery constructs, or portionsthereof. The cell is preferably selected for its ability to express highconcentrations of the delivery construct to facilitate purification ofthe protein. In certain embodiments, the cell is a prokaryotic cell, forexample, E. coli. As described in the examples, the delivery constructsare properly folded and comprise the appropriate disulfide linkages whenexpressed in E. coli.

In other embodiments, the cell is a eukaryotic cell. Useful eukaryoticcells include yeast and mammalian cells. Any mammalian cell known by oneof skill in the art to be useful for expressing a recombinantpolypeptide, without limitation, can be used to express the deliveryconstructs. For example, Chinese hamster ovary (CHO) cells can be usedto express the delivery constructs.

5.7. Compositions Comprising Delivery Constructs

The delivery constructs of the invention can be formulated ascompositions. The compositions are generally formulated appropriatelyfor the immediate use intended for the delivery construct. For example,if the delivery construct is not to be administered immediately, thedelivery construct can be formulated in a composition suitable forstorage. One such composition is a lyophilized preparation of thedelivery construct together with a suitable stabilizer. Alternatively,the delivery construct composition can be formulated for storage in asolution with one or more suitable stabilizers. Any such stabilizerknown to one of skill in the art without limitation can be used. Forexample, stabilizers suitable for lyophilized preparations include, butare not limited to, sugars, salts, surfactants, proteins, chaotropicagents, lipids, and amino acids. Stabilizers suitable for liquidpreparations include, but are not limited to, sugars, salts,surfactants, proteins, chaotropic agents, lipids, and amino acids.Specific stabilizers than can be used in the compositions include, butare not limited to, trehalose, serum albumin, phosphatidylcholine,lecithin, and arginine. Other compounds, compositions, and methods forstabilizing a lyophilized or liquid preparation of the deliveryconstructs may be found, for example, in U.S. Pat. Nos. 6,573,237,6,525,102, 6,391,296, 6,255,284, 6,133,229, 6,007,791, 5,997,856, and5,917,021.

Further, the delivery construct compositions of the invention can beformulated for administration to a subject. Such vaccine compositionsgenerally comprise one or more delivery constructs of the invention anda pharmaceutically acceptable excipient, diluent, carrier, or vehicle.Any such pharmaceutically acceptable excipient, diluent, carrier, orvehicle known to one of skill in the art without limitation can be used.Examples of a suitable excipient, diluent, carrier, or vehicle can befound in Remington's Pharmaceutical Sciences, 21st Ed. 2005, MackPublishing Co., Easton.

In certain embodiments, the delivery construct compositions areformulated for oral administration. In such embodiments, thecompositions are formulated to protect the delivery construct from acidand/or enzymatic degradation in the stomach. Upon passage to the neutralto alkaline environment of the duodenum, the delivery construct thencontacts a mucous membrane and is transported across the polarizedepithelial membrane. The delivery constructs may be formulated in suchcompositions by any method known by one of skill in the art, withoutlimitation.

In certain embodiments, the oral formulation comprises a deliveryconstruct and one or more compounds that can protect the deliveryconstruct while it is in the stomach. For example, the protectivecompound should be able to prevent acid and/or enzymatic hydrolysis ofthe delivery construct. In certain embodiments, the oral formulationcomprises a delivery construct and one or more compounds that canfacilitate transit of the construct from the stomach to the smallintestine. In certain embodiments, the one or more compounds that canprotect the delivery construct from degradation in the stomach can alsofacilitate transit of the construct from the stomach to the smallintestine. Preferably, the oral formulation comprises one or morecompounds that can protect the delivery construct from degradation inthe stomach and facilitate transit of the construct from the stomach tothe small intestine. For example, inclusion of sodium bicarbonate can beuseful in facilitating the rapid movement of intra-gastric deliveredmaterials from the stomach to the duodenum as described in Mrsny et al.,1999, Vaccine 17:1425-1433.

Other methods for formulating compositions so that the deliveryconstructs can pass through the stomach and contact polarized epithelialmembranes in the small intestine include, but are not limited to,enteric-coating technologies as described in DeYoung, 1989, Int JPancreatol. 5 Suppl:31-6, and the methods provided in U.S. Pat. Nos.6,613,332, 6,174,529, 6,086,918, 5,922,680, and 5,807,832.

5.7.1. Kits Comprising Compositions

In yet another aspect, the invention provides a kit that comprises acomposition of the invention. In certain embodiments, the kit furthercomprises instructions that direct administration of the composition toa mucous membrane of the subject to whom the composition isadministered. In certain embodiments, the kit further comprisesinstructions that direct oral administration of the composition to thesubject to whom the composition is administered.

In certain embodiments, the kit comprises a composition of the inventionin more or more containers. In certain embodiments, the composition canbe in a unit dosage form, e.g., a tablet, lozenge, capsule, etc. Incertain embodiments, the composition can be provided in or with a devicefor administering the composition, such as, for example, a deviceconfigured to administer a single-unit dose of the composition, e.g., aninhaler.

5.8. Making and Testing Delivery Constructs

The delivery constructs of the invention are preferably producedrecombinantly, as described below. However, the delivery constructs mayalso be produced by chemical synthesis using methods known to those ofskill in the art.

5.8.1. Manufacture of Delivery Constructs

Methods for expressing and purifying the delivery constructs of theinvention are described extensively in the examples below. Generally,the methods rely on introduction of an expression vector encoding thedelivery construct to a cell that can express the delivery constructfrom the vector. The delivery construct can then be purified foradministration to a subject.

5.8.2. Testing Delivery Constructs

Having selected the domains of the delivery construct, the function ofthese domains, and of the delivery constructs as a whole, can beroutinely tested to ensure that the constructs can deliver amacromolecule across mucous membranes of a subject free from theremainder of the construct, For example, the delivery constructs can betested for cell recognition, transcytosis and cleavage using routineassays. The entire chimeric protein can be tested, or, the function ofvarious domains can be tested by substituting them for native domains ofthe wild-type toxin.

5.8.2.1. Receptor binding/Cell recognition

Receptor binding domain function can be tested by monitoring thedelivery construct's ability to bind to the target receptor. Suchtesting can be accomplished using cell-based assays, with the targetreceptor present on a cell surface, or in cell-free assays. For example,delivery construct binding to a target can be assessed with affinitychromatography. The construct can be attached to a matrix in an affinitycolumn, and binding of the receptor to the matrix detected, or viceversa. Alternatively, if antibodies have been identified that bind toeither the receptor binding domain or its cognate receptor, theantibodies can be used, for example, to detect the receptor bindingdomain in the delivery construct by immunoassay, or in a competitionassay for the cognate receptor. An exemplary cell-based assay thatdetects delivery construct binding to receptors on cells compriseslabeling the construct and detecting its binding to cells by, e.g.,fluorescent cell sorting, autoradiography, etc.

5.8.2.2. Transcytosis

The function of the transcytosis domain can be tested as a function ofthe delivery construct's ability to pass through an epithelial membrane.Because transcytosis first requires binding to the cell, these assayscan also be used to assess the function of the cell recognition domain.

The delivery construct's transcytosis activity can be tested by anymethod known by one of skill in the art, without limitation. In certainembodiments, transcytosis activity can be tested by assessing theability of a delivery construct to enter a non-polarized cell to whichit binds. Without intending to be bound to any particular theory ormechanism of action, it is believed that the same property that allows atranscytosis domain to pass through a polarized epithelial cell alsoallows molecules bearing the transcytosis domain to enter non-polarizedcells. Thus, the delivery construct's ability to enter the cell can beassessed, for example, by detecting the physical presence of theconstruct in the interior of the cell. For example, the deliveryconstruct can be labeled with, for example, a fluorescent marker, andthe delivery construct exposed to the cell. Then, the cells can bewashed, removing any delivery construct that has not entered the cell,and the amount of label remaining determined. Detecting the label inthis traction indicates that the delivery construct has entered thecell.

In other embodiments, the delivery construct's transcytosis ability canbe tested by assessing the delivery construct's ability to pass througha polarized epithelial cell. For example, the delivery construct can belabeled with, for example, a fluorescent marker and contacted to theapical membranes of a layer of epithelial cells. Fluorescence detectedon the basal-lateral side of the membrane formed by the epithelial cellsindicates that the transcytosis domain is functioning properly.

5.8.2.3. Cleavable Linker Cleavage

The function of the cleavable linker can generally be tested in acleavage assay. Any suitable cleavage assay known by one of skill in theart, without limitation, can be used to test the cleavable linkers. Bothcell-based and cell-free assays can be used to test the ability of anenzyme to cleave the cleavable linkers.

An exemplary cell-free assay for testing cleavage of cleavable linkerscomprises preparing extracts of polarized epithelial cells and exposinga labeled delivery construct bearing a cleavable linker to the fractionof the extract that corresponds to membrane-associated enzymes. In suchassays, the label can be attached to either the macromolecule to bedelivered or to the remainder of the delivery construct. Among theseenzymes are cleavage enzymes found near the basal-lateral membrane of apolarized epithelial cell, as described above. Cleavage can be detected,for example, by binding the delivery construct with, for example, anantibody and washing off unbound molecules. If label is attached to themacromolecule to be delivered, then little or no label should beobserved on the molecule bound to the antibodies. Alternatively, thebinding agent used in the assay can be specific for the macromolecule,and the remainder of the construct can be labeled. In either case,cleavage can be assessed.

Cleavage can also be tested using cell-based assays that test cleavageby polarized epithelial cells assembled into membranes. For example, alabeled delivery construct, or portion of a delivery constructcomprising the cleavable linker, can be contacted to either the apicalor basolateral side of a monolayer of suitable epithelial cells, suchas, for example, Coco-2 cells, under conditions that permit cleavage ofthe linker. Cleavage can be detected by detecting the presence orabsence of the label using a reagent that specifically binds thedelivery construct, or portion thereof. For example, an antibodyspecific for the delivery construct can be used to bind a deliveryconstruct comprising a label distal to the cleavable linker in relationto the portion of the delivery construct bound by the antibody. Cleavagecan then be assessed by detecting the presence of the label on moleculesbound to the antibody. If cleavage has occurred, little or no labelshould be observed on the molecules bound to the antibody. By performingsuch experiments, enzymes that preferentially cleave at the basolateralmembrane rather than the apical membrane can be identified, and,further, the ability of such enzymes to cleave the cleavable linker in adelivery construct can be confirmed.

Further, cleavage can also be tested using a fluorescence reporter assayas described in U.S. Pat. No. 6,759,207. Briefly, in such assays, thefluorescence reporter is contacted to the basolateral side of amonolayer of suitable epithelial cells under conditions that allow thecleaving enzyme to cleave the reporter. Cleavage of the reporter changesthe structure of the fluorescence reporter, changing it from anon-fluorescent configuration to a fluorescent configuration. The amountof fluorescence observed indicates the activity of the cleaving enzymepresent at the basolateral membrane.

Further, cleavage can also be tested using an intra-molecularly quenchedmolecular probe, such as those described in U.S. Pat. No. 6,592,847.Such probes generally comprise a fluorescent moiety that emits photonswhen excited with light of appropriate wavelength and a quencher moietythat absorbs such photons when in close proximity to the fluorescentmoiety. Cleavage of the probe separates the quenching moiety from thefluorescent moiety, such that fluorescence can be detected, therebyindicating that cleavage has occurred. Thus, such probes can be used toidentify and assess cleavage by particular cleaving enzymes bycontacting the basolateral side of a monolayer of suitable epithelialcells with the probe under conditions that allow the cleaving enzyme tocleave the probe. The amount of fluorescence observed indicates theactivity of the cleaving enzyme being tested.

6. EXAMPLES

The following examples merely illustrate the invention, and are notintended to limit the invention in any way.

6.1. Construction of a Delivery Construct

Five exemplary delivery construct expression vectors for delivering ratgrowth hormone (rGH) were constructed according to the followingprotocol. First, the rGH gene was amplified by PCR, incorporatingrestriction enzymes pairs of NdeI and EcoRI, PstI and PstI, AgeI andEcoRI, or PstI and EcoRI sites at two ends of the PCR products. Afterrestriction enzyme digestion, the PCR products were cloned intopPE64-PstI-Δ553, which was digested with the corresponding restrictionenzyme pairs. The resulting constructs were named aspPE-RGH(NdeI-EcoRI), pntPE-RGH(PstI), pntPE-RGH(AgeI-EcoRI), andpPE-RGH(PstI-EcoRI). These constructs thus comprise sequences encodingDomains I and II of ntPE (amino acids 26-372 as shown in FIG. 3) and rGH(Accession No. P01244; see Seeburg et al., 1977, Nature 270:486-494 andPage et al., 1981, Nucleic Acids Res. 9:2087-2104), and are also taggedwith a 6-His motif at the N-terminus of the polypeptide to facilitatepurification. The final plasmids were verified by restriction enzymedigestions and DNA sequencing.

Expression vectors comprising cleavable linkers were constructed byintroducing sequences encoding the appropriate amino acid sequence. Todo so, oligonucleotides that encode sequences complementary toappropriate restriction sites and one of the following amino acidsequences were synthesized, then ligated into an expression vectorprepared as described above between the ntPE sequences and the rGHsequences. For Delivery Construct 1, the cleavable linker sequence wasRQPRGGL. For Delivery Construct 2, the cleavable linker sequence wasGGLRQPR. For Delivery Construct 3, the cleavable linker sequence wasRQPREGR. For Delivery Construct 4, the cleavable linker sequence wasRQPRVGR. For Delivery Construct 5, the cleavable linker sequence wasRQPRARR.

To separate rGH from PE protein in the event that the fusion protein istaken up by antigen presenting cells, a protease furin site was alsoinserted between the cleavable linker and rGH. To do so, constructscontaining a sequence encoding the furin site with the five differentcleavable linkers were made. Oligonucleotide sequences for the fivecleavable linkers and a furin clip site are shown in Table 3 below. Eachof the oligo duplexes was inserted into PstI site ofpPE-RGH(PstI-EcoRI). The final constructs, named as pPE-RGH-F1,pPE-RGH-F2, pPE-RGH-F3, pPE-RGH-F4, pPE-RGH-F5 were confirmed byrestriction enzyme digestion and DNA sequencing.

TABLE 3 Oligonucleotide pairs for introducing a furin cleavage site andprotease cleavage site pPE-RGH-1 1 F:AACTGCAGCGCCAGCCTCGAGGAGGATTACTGCAGAA (SEQ ID NO: 24) 1 R:TTCTGCAGTAATCCTCCTCGAGGCTGGCGCTGCAGTT (SEQ ID NO: 25) pPE-RGH-2 2 F:AACTGCAGGGAGGCTTACGCCAGCCTCGACTGCAGAA (SEQ ID NO: 26) 2 R:TTCTGCAGTCGAGGCTGGCGTAAGCCTCCCTGCAGTT (SEQ ID NO: 27) pPE-RGH-3 3 F:AACTGCAGCGCCAGCCTCGAGAGGGCCGTCTGCAGAA (SEQ ID NO: 28) 3 R:TTCTGCAGACGGCCCTCTCGAGGCTGGCGCTGCAGTT (SEQ ID NO: 29) pPE-RGH-4 4 F:AACTGCAGCGCCAGCCTCGAGTCGGCCGTCTGCAGAA (SEQ ID NO: 30) 4 R:TTCTGCAGACGGCCGACTCGAGGCTGGCGCTGCAGTT (SEQ ID NO: 31) pPE-RGH-5 5 F:AACTGCAGCGCCAGCCTCGAGCACGTCGTCTGCAGAA (SEQ ID NO: 32) 5 R:TTCTGCAGACGACGTGCTCGAGGCTGGCGCTGCAGTT (SEQ ID NO: 33)

6.2. Expression of Delivery Constructs

E. coli BL21(DE3) pLysS competent cells (Novagen, Madison, Wis.) weretransformed using a standard heat-shock method in the presence of theappropriate plasmid to generate ntPE-rat Growth Hormone (rGH) expressioncells, selected on ampicillin-containing media, and isolated and grownin Luria-Bertani broth (Difco; Becton Dickinson, Franklin Lakes, N.J.)with antibiotic, then induced for protein expression by the addition of1 mM isopropyl-D-thiogalactopyranoside (IPTG) at OD 0.6. Two hoursfollowing IPTG induction, cells were harvested by centrifugation at5,000 rpm for 10 min. Inclusion bodies were isolated following celllysis and proteins were solubilized in the buffer containing 100 mMTris_HCl (pH 8.0), 2 mM EDTA, 6 M guanidine HCl, and 65 mMdithiothreitol. Solubilized His ntPE-rGH is refolded in the presence of0.1 M Tris, pH=7.4, 500 mM L-arginine, 0.9 mM GSSG, 2 mM EDTA. Therefolded proteins were purified by Q sepharose Ion Exchange and Superdex200 Gel Filtration chromatography (Amersham Biosciences, Inc., Sweden).The purity of proteins was assessed by SDS-PAGE and analytic HPLC(Agilent, Inc. Palo Alto, Calif.).

6.3. Characterization of a Delivery Construct

The following procedures can be used to assess proper refolding of adelivery construct. The protein refolding process is monitored bymeasuring, e.g., Delivery Construct 1 binding activity with ntPE bindingreceptor, CD 91 receptors, and rGH binding proteins on a Biacore SPRinstrument (Biacore, Sweden) according to the manufacturer'sinstructions. Proper refolding of other macromolecules in exemplaryconstructs can be tested in similar binding assays with appropriatebinding agents. By testing such binding affinities, the skilled artisancan assess the proper folding of each portion of the delivery construct.

6.4. Delivery Construct Cleavage Assays

This example describes experiments performed to identify and verifyenzymes that can be used to cleave the cleavable linkers of the deliveryconstructs described herein. First, Caco-2 (ATCC Accession No. HTB-37)cells in passage 21 were obtained from American Type Culture Collection(Manassas, Va.). Human tracheal epithelial (HTE) cells were obtainedfrom J. Whiddecombe of the Department of Physiology at the University ofCalifornia, Davis Medical School. Caco-2 cells were routinely grown on75 cm² plastic culture flasks (Becton Dickinson, Franklin Lakes, N.J.)in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycinat 37° C. in a 5% CO₂/95% air atmosphere. HTE cells were grown asdescribed in Yamaya et al., 1992, Am J Physiol 262(6 Pt 1):L713-24.

To identify suitable cleavable linkers, HTE or Caco-2 cells were seededat a density of 5×10⁴ cells/cm² onto 24-well collagen-coatedpolycarbonate transwell filters (Corning, Acton, Mass.) for 12-14 days.Confluent monolayers achieved a transepithelial resistance (TER) of >500ohm·cm², as measured using an EVOM epithelial voltohmmeter and STX2electrode (World Precision Instruments, Sarasota, Fla.). To determinespecific enzyme activity, substrates specific for the tested peptidase(500 μM or 1 mM substrate in 250 μl DMEM without FBS or antibiotics)were added to either the apical (AP) or basolateral (BL) side of themonolayers. Peptidase substrates were obtained from Calbiochem, Inc.(Division of EMD Biosciences, Inc., San Diego, Calif.). Cells wereincubated for 2 hrs at 37° C. in a 5% CO₂/95% air atmosphere. Both theapical and basolateral media was then measured for its specific enzymeactivity according to the manufacturer's instruction. Cleavage wasassessed by detecting fluorescence of the substrates, which reflectscleavage because it separates of the quenching agent from thefluorescent agent present on the substrate, which separation allowsfluorescence to be detected.

Table 4 presents a summary of the results of these assays using HTEcells, while Table 5 presents a summary of the results of these assaysusing Caco-2 cells. For all results, baseline control values weresubtracted from substrate values before percentages were determined andtests were performed at least in duplicate. The percentages presented inthe tables represent the percent increase observed in assay in theapical or basolateral media, which depends on which side of the membraneexhibits higher peptidase activity. It should be noted that, even whensubstrate was added to the media on the apical side of the membrane,peptidase activity can be observed on the basolateral side of themembrane because of diffusion of the substrate across the membrane.

TABLE 4 AP BL Peptidase tested in % % A405 A405 HTE cells AP > BL BL >AP nm nm AP-500 uM Cathepsin B I 86% 0.37 0.20 BL-500 uM Cathepsin B I64% 0.35 0.21 AP-500 uM Cathepsin G I 882%  0.04 0.004 BL-500 uMCathepsin G I  6% 0.02 0.02 AP-500 uM Cathepsin G II 371%  0.02 0.01BL-500 uM Cathepsin G II  0%  0% 0.02 0.02 AP-500 uM Cathepsin G III 11%0.02 0.02 BL-500 uM Cathepsin G III 400%  0.01 0.03 AP-500 uMChymotrypsin I 74% 0.04 0.02 BL-500 uM Chymotrypsin I 36% 0.03 0.04AP-500 uM Elastase I 49% 0.05 0.03 BL-500 uM Elastase I 23% 0.02 0.03AP-500 uM Elastase II 43% 0.29 0.20 BL-500 uM Elastase II 31% 0.18 0.13AP-500 uM Elastase III 89% 0.04 0.02 BL-500 uM Elastase III 967%  0.030.003 AP-500 uM Elastase IV 84% 0.35 0.19 BL-500 uM Elastase IV 65% 0.230.14 AP-500 uM Elastase VIII 529%  0.16 0.03 BL-500 uM Elastase VIII181%  0.09 0.03 AP-500 uM Papain 57% 0.02 0.02 BL-500 uM Papain  5% 0.030.03 AP-500 uM Subtilisin A I  9% 0.02 0.02 BL-500 uM Subtilisin A I3000%  0.001 0.03 AP-500 uM Subtilisin A II 21% 0.02 0.02 BL-500 uMSubtilisin A II 55% 0.01 0.02 AP-500 uM Thrombin I 42% 0.15 0.11 BL-500uM Thrombin I 15% 0.09 0.10 AP-500 uM Thrombin II 445%  0.40 0.07 BL-500uM Thrombin II 741%  0.41 0.05 AP-500 uM Urokinase I  8% 0.11 0.10BL-500 uM Urokinase I  4% 0.13 0.13 AP-1 mM Cathepsin B I 17% 0.18 0.15BL-1 mM Cathepsin B I 42% 0.24 0.17 AP-1 mM Cathepsin G I 114%  0.050.02 BL-1 mM Cathepsin G I 47% 0.02 0.03 AP-1 mM Cathepsin G II 138% 0.05 0.02 BL-1 mM Cathepsin G II 19% 0.03 0.03 AP-1 mM Cathepsin G III225%  0.07 0.02 BL-1 mM Cathepsin G III 54% 0.02 0.03 AP-1 mMChymotrypsin I 35% 0.06 0.04 BL-1 mM Chymotrypsin I 90% 0.04 0.07 AP-1mM Elastase I 108%  0.02 0.03 BL-1 mM Elastase I 864%  0.01 0.09 AP-1 mMElastase II 62% 0.28 0.17 BL-1 mM Elastase II 42% 0.33 0.23 AP-1 mMElastase III 318%  0.02 0.01 BL-1 mM Elastase III 131%  0.04 0.02 AP-1mM Elastase IV 94% 0.41 0.21 BL-1 mM Elastase IV 61% 0.30 0.19 AP-1 mMElastase VIII 233%  0.14 0.04 BL-1 mM Elastase VIII 41% 0.06 0.05 AP-1mM Papain 424%  0.02 0.004 BL-1 mM Papain 141%  0.02 0.01 AP-1 mMSubtilisin A I 18% 0.03 0.03 BL-1 mM Subtilisin A I 290%  0.01 0.04 AP-1mM Subtilisin A II 17% 0.03 0.02 BL-1 mM Subtilisin A II 318%  0.01 0.04AP-1 mM Thrombin I 27% 0.17 0.14 BL-1 mM Thrombin I 28% 0.17 0.13 AP-1mM Thrombin II 20% 0.12 0.10 BL-1 mM Thrombin II 13% 0.05 0.06 AP-1 mMUrokinase I 14% 0.19 0.21 BL-1 mM Urokinase I  4% 0.21 0.20

TABLE 5 AP BL Peptidase tested in % % A405 A405 Caco-2 cells AP > BLBL > AP nm nm AP-500 uM Cathepsin B I 150% 0.14 0.06 BL-500 uM CathepsinB I  34% 0.17 0.12 AP-10 mM Cathepsin G I 195% 0.31 0.10 BL-10 mMCathepsin G I 145% 0.42 1.03 AP-500 uM Cathepsin G III  35% 0.014 0.01BL-500 uM Cathepsin G III 185% 0.03 0.01 AP-500 uM Cathepsin G I 232%0.05 0.01 BL-500 uM Cathepsin G I 1709%  0.01 0.15 AP-500 uM Cathepsin GII 3900%  0.01 0.0003 BL-500 uM Cathepsin G II 1330%  0.003 0.04 AP-500uM Chymotrypsin I 342% 0.01 0.04 BL-500 uM Chymotrypsin I 403% 0.03 0.16AP-500 uM Elastase I  73% 0.01 0.01 BL-500 uM Elastase I 295% 0.04 0.01AP-500 uM Elastase II  59% 0.12 0.07 BL-500 uM Elastase II  89% 0.010.02 AP-500 uM Elastase III  85% 0.01 0.07 BL-500 uM Elastase III 320%0.003 0.01 AP-500 uM Elastase IV  32% 0.11 0.08 BL-500 uM Elastase IV 12% 0.02 0.02 AP-500 uM Elastase VIII  16% 0.02 0.02 BL-500 uM ElastaseVIII 115% 0.01 0.02 AP-500 uM Papain  19% 0.018 0.02 BL-500 uM Papain339% 0.07 0.02 AP-500 uM Subtilisin A I ***23%  — 0.05 BL-500 uMSubtilisin A I ***94%  — 0.20 AP-500 uM Subtilisin A II N/A — — BL-500uM Subtilisin A II ***11%  — 0.02 AP-500 uM Thrombin I  81% 0.04 0.02BL-500 uM Thrombin I 254% 0.01 0.04 AP-Thrombin II 500 uM  42% 0.08 0.06BL-Thrombin II 500 uM  62% 0.09 0.06 AP-500 uM Urokinase I 111% 0.120.06 BL-500 uM Urokinase I 1044%  0.005 0.05 AP-1 mM Cathepsin B I 109%0.27 0.13 BL-1 mM Cathepsin B I  58% 0.12 0.2 AP-20 mM Cathepsin G I129% 0.10 0.23 BL-20 mM Cathepsin G I 540% 0.11 0.70 AP-1 mM Cathepsin GIII  37% 0.01 0.01 BL-1 mM Cathepsin G III 103% 0.07 0.03 AP-1 mMCathepsin G I 107% 0.08 0.04 BL-1 mM Cathepsin G I 144% 0.12 0.05 AP-1mM Cathepsin G II  11% 0.05 0.06 BL-1 mM Cathepsin G II 7850%  0.00 0.04AP-1 mM Chymotrypsin I 107% 0.08 0.04 BL-1 mM Chymotrypsin I 288% 0.020.07 AP-1 mM Elastase I 217% 0.03 0.001 BL-1 mM Elastase I 880% 0.0030.02 AP-1 mM Elastase II  27% 0.17 0.14 BL-1 mM Elastase II  34% 0.020.03 AP-1 mM Elastase III 192% 0.02 0.01 BL-1 mM Elastase III  77% 0.020.01 AP-1 mM Elastase IV  42% 0.16 0.11 BL-1 mM Elastase IV  10% 0.040.05 AP-1 mM Elastase VIII  70% 0.05 0.03 BL-1 mM Elastase VIII 332%0.11 0.03 AP-1 mM Papain  61% 0.02 0.01 BL-1 mM Papain  0% 0.005 0.005AP-1 mM Subtilisin A I ***61%  — 0.13 BL-1 mM Subtilisin A I ***44%  —0.09 AP-1 mM Subtilisin A II N/A — — BL-1 mM Subtilisin A II N/A — —AP-1 mM Thrombin I 420% 0.11 0.02 BL-1 mM Thrombin I 3400%  0.005 0.16AP-Thrombin II 1 mM 163% 0.14 0.05 BL-Thrombin II 1 mM  29% 0.11 0.09AP-1 mM Urokinase I  57% 0.17 0.11 BL-1 mM Urokinase I 230% 0.05 0.15***denotes % over baseline control only “—” denotes values belowbaseline control

6.5. Delivery of an Exemplary Macromolecule—Green Fluorescent Protein

The following example describes experiments performed to assess thetranscytosis of an exemplary delivery construct for delivering greenfluorescent protein (“GFP”) across a mouse epithelial membrane. It isnoted that this exemplary delivery construct does not comprise acleavable linker; however, the presence or absence of the cleavablelinker should not affect transcytosis of the delivery construct.

Briefly, a nt-PE-GFP construct was applied to the trachea ofanesthetized female balb/c mice which were approximately 8 weeks of age.The mice were anesthetized with inhaled isoflurane and the trachea wasexposed. A small hole was made on the trachea to allow application ofour GFP material. In our experiments, 100 μg of GFP alone or ntPE-GFPwas used, respectively. The GFP material was slowly dripped directlyonto the exposed trachea in a 100 μl volume. After 15 minutes, the micewere euthanized by CO₂ asphyxiation. The trachea was removed and frozenin OCT (cat#25608-930-Tissue Tek) using biopsy cryomolds(cat#4565-Tissue Tek). The samples were sectioned onto slides andvisualized by fluorescence microscopy (Nikon model Eclipse E400).

Micrographs of the epithelial sections are presented as FIGS. 1A-1C.FIG. 1A shows the nt-PE-GFP construct adhering strongly to the apicalsurface of the trachea epithelium. FIG. 1B shows transcytosis of thent-PE-GFP construct across the trachea epithelium. FIG. 1C shows releaseof the nt-PE-GFP construct from the basolateral side of the tracheaepithelium. FIG. 1D presents a micrograph of a negative control, atracheal epithelial section from a mouse contacted with GFP alone. Thetissues exposed for 15 minutes to obtain these micrographs.

The micrographs demonstrate that nt-PE-GFP interacts strongly withreceptors on the apical surface of mouse tracheal epithelium,transcytoses across such epithelial tissue, and releases from thebasolateral surface of the mouse tracheal epithelium.

In addition, plasma concentrations of the nt-PE-GFP construct weredetermined following administration of the delivery construct using anELISA assay as described in Example 6.6.1, below. Serum samples weretaken from anesthetized mice that had received intranasal administrationof 100 μg of the nt-PE-GFP delivery construct every 30 minutes followingadministration. FIG. 2 presents the results of this experiment,demonstrating that peak plasma levels of the delivery construct reachedbetween 500-900 ng/ml, indicating that the delivery construct displayedapproximately 22% bioavailability following intranasal administration.

6.6. Detection of Growth Hormone Protein in Tissue by HistologicalExamination

This example describes histological detection in tissues of arepresentative macromolecule for delivery, growth hormone. Followingadministration of a delivery construct, animals are euthanized by CO₂asphyxiation and exanguinated by cardiac puncture. Specific tissues(lymph nodes, trachea, brain, spleen liver, GI tract) are removed,briefly rinsed in PBS to remove any residual blood and frozen in OCT.Sections (5 microns thick) are placed onto slides. Slides are fixed inacetone for 10 min and rinsed with PBS. Slides are incubated with 3%peroxidase for 5 min. Slides are then blocked with protein for anadditional 5 min. Primary growth hormone antibody is incubated ontoslides for 30 min at a 1:100 dilution followed by PBS washes.Biotin-labeled secondary antibody is then incubated for approximately 15minutes followed by PBS washes. Streptavidin HRP label is incubated ontoslides for 15 min followed by PBS washes. HRP Chromagen is applied for 5min followed by several rinses in distilled H₂0. Finally, the slides arecounterstained with hematoxylin for 1 min, coverslipped, and examinedfor the presence of GH.

6.7. Transport and Cleavage of an Exemplary Delivery Construct in an inVitro System

This example describes transport and cleavage of an exemplary deliveryconstruct, Delivery Construct 2, comprising rat growth hormone (rGH) inan in vitro system using human tracheal epithelial cells.

6.7.1. Growth of Human Tracheal Epithelial Cells

Human tracheal epithelial (HTE) cells were isolated from tracheas aspreviously described and cultured on semi-permeable filter systems (0.45um pore size; Corning, Acton, Mass.) coated with human placentalcollagen. See Yamaya et al., 1992, Am J Physiol, 262:L713-24 and Sachset al., 2003, In Vitro Cell Dev Biol Anim, 39:56-62. Cell sheets wereused at >10 days following plating, at which time they had atransepithelial resistance (TER) of >100 Ω·ohms·cm² as measured with a“chopstick” voltohmeter (Millicell ERS, Manassas, Va.).

Caco-2 cells in passage 21 were obtained from American Type CultureCollection (Manassas, Va.). Cells were routinely grown on 75 cm² plasticculture flasks (Becton Dickinson, Franklin Lakes, N.J.) in DMEMcontaining 10% fetal bovine serum (FBS) and 1% penicillin-streptomycinat 37° C. in a 5% CO₂/95% air atmosphere. For the transport and cleavagestudies, Caco-2 cells were seeded at a density of 5×10⁴ cells/cm² onto24-well collagen-coated polycarbonate transwell filters (Corning, ActonMass.) for 12-14 days. Confluent monolayers achieved a transepithelialresistance (TER) of >500 ohm/cm², as measured using the EVOM and STX2electrode (World Precision Instruments).

6.7.2. Transport and Cleavage Assays

To determine transport and cleavage activity, Delivery Constructs 1 and2 proteins (10 μg in 100 μl DMEM without phenol red, FBS or antibiotics)were added to the apical side of the epithelial monolayer. Cells wereincubated for 4 hrs at 37° C. in a 5% CO₂/95% air atmosphere. Both theapical and basolateral media was then assayed for its transport andcleavage activity by testing for the presence of rGH cleaved from thedelivery construct by western blot analysis, as described below. As acontrol, 10 μg of dextran fluorescein was also added to the apical wellsin order to check for leakage.

Following the 4 hour incubation described above, apical and basolateralmedia samples were precipitated by the addition of trichloroacetic acid(TCA). The amount of TCA added to each sample was twenty percentrelative to the sample volume. Each sample was vortexed and placed onice for 30 minutes. After samples this incubation on ice, samples werecentrifuged at 14,000 RPM for 10 minutes. Next, the supernatants fromeach sample were aspirated and tubes containing the remaining pelletwere left open to air dry. 4 μl 0.2M NaOH was added to each pellet. Fiveminutes after the addition of NaOH, pellets were resuspended in 36 μl 8MUrea.

Next, 10 μl Sample Buffer containing DTT (Invitrogen NP0007, NP0009) wasadded to each sample. Samples were then placed on a 100° C. heatingblock for 5 minutes. Half (19 μl) of each sample was then loaded on to a4%-12% Tris Bis Gel (Invitrogen NP3022BOX). For controls, recombinantrat GH (RDI R0125) and Delivery Construct 1 or Delivery Construct 2proteins were also loaded directly into the gel. Electrophoresis was at150V for 30 minutes. From the gel, samples were transferred ontonitrocellulose at 30V for 1 hour.

The Blocking Solution, Antibody Diluents, and Antibody Wash Solutionfrom Invitrogen's WesternBreeze were used in subsequent steps. Thenitrocellulose membrane was placed in blocking buffer and incubated at4° C., overnight. The membrane was washed 3 times for 3 minutes eachtime. The membrane was then incubated at RT with 10 ml of 1:2000 rabbitanti growth hormone (RDI RDIRtGHabr). After one hour, the membrane wasagain rinsed 3 times for 3 minutes each. Membrane was incubated for 1hour at room temperature with 10 ml of goat anti rabbit IgG AP (Pierce31340) at 1:5000. The membrane was rinsed 3 times for 3 minutes perwash. 5 μl of substrate (Pierce 34042) was added to the membrane. Aftercolor development reached desired intensity, reaction was halted by theremoval of substrate and the addition of purified water. Finally, themembrane was washed in purified water for 30 minutes and air dried.

Results of the Western Blot analysis are presented in FIG. 4. As seen inFIG. 4, media from the basolateral side of the epithelial cell layercontained protein consistent with cleaved rGH separated from theremainder of Delivery Construct 2. In contrast, media from the apicalside of the epithelial cell layer contained largely intact deliveryconstruct. Thus, application of Delivery Construct 2 to the apical sideof the human epithelial cell membrane resulted in both transport to thebasolateral side of the membrane and proper cleavage of the construct asshown by release of rGH detectable with anti-r(1H antibody and of properapparent molecular weight. Similar results were also observed forDelivery Construct 1 (data not shown).

6.8. Delivery of an Exemplary Macromolecule in an In Vivo System

This example describes use of exemplary Delivery Construct 2 in a mousemodel, showing effective transport and cleavage of the deliveryconstruct in vivo and the bioactivity of the macromolecule delivered byDelivery Construct 2, rGH.

6.8.1. Administration of a Delivery Construct Comprising Rat GrowthHormone

Using an animal feeding needle, 100 μg of Delivery Construct 2 (in 250μl total volume) was orally delivered to female BALB/c mice, 5-6 weeksof age (Charles River Laboratories, Wilmington, Mass.). DeliveryConstruct 2 was diluted in 1 mg/ml bovine serum albumin (BSA) andphosphate buffered saline (PBS). As a positive control, control micewere subcutaneously (SC) injected on their dorsal side with 30 μg ofrecombinant rat growth hormone (rGH) diluted in PBS (100 μl totalvolume). At specific times after oral gavage and SC administration, micewere euthanized by CO₂ asphyxiation and exsanguinated. Whole blood andliver were collected and analyzed as described below. Because in thedifference in molecular weights between Delivery Construct 2 and rGH,essentially the same number of rGH molecules were administered in bothroutes.

6.8.2. Pharmacokinetics of an Exemplary Macromolecule Administered witha Delivery Construct in an In Vivo System

To assess the pharmacokinetics of an exemplary macromolecule deliveredwith a delivery construct, ELISA assays were used to measure serumconcentrations of rGH at defined timepoints following administration.The serum concentration data thus obtained was used to compare thepharmacokinetics of rGH administered with Delivery Construct 2 to thoseobserved with conventional subcutaneous administration. The ELISA assayswere performed as follows.

Costar 9018 E.I.A./R.I.A. 96-well plates were coated overnight with 200ng/well of goat anti-rGH (Diagnostics Systems Laboratories, Cat. No.R01235) in 0.2M NaHCO₃—Na₂CO₃, pH 9.4. Each 96-well plate was washedfour times with PBS containing 0.05% Tween 20-0.01% thimerosal (washbuffer); blocked for 1 h with 200 μl/well of PBS/Tween 20 containing0.5% BSA-0.01% thimerosal (assay buffer). The standard curve wasprepared using recombinant rat GH (Diagnostics Systems Laboratories,Cat. No. R01205) diluted in assay buffer (PBST-0.5% BSA). The firstpoint of the standard curve was prepared by adding 50 μl recombinant ratGH to 10 ml assay buffer (1:200), vortexed, and moved 200 μl to 800 μlassay buffer (1:5). For each plate, 0.5 ml was moved to 0.5 ml assaybuffer by doing 1:2 serial dilutions for all the subsequent points. The10 points of the standard curve are: 100, 50, 25, 12.5, 6.25, 3.125,1.56, 0.78, 0.39 and 0.195 ng/well. Samples were diluted at 1:10 withassay buffer, loaded 100 μl/well in triplicates onto a 96-well plate,and incubated overnight. Each 96-well plate was then washed four timeswith wash buffer, loaded 100 μl/well of 2^(nd) Ab (rabbit anti-rGH, CellSciences, Cat. No. PAAC1) at 1:300 in assay buffer (PBST-0.5% BSA) andincubate at RT for four hours. Each 96-well plate was then washed fourtimes with wash buffer, loaded 100 μl/well of 3rd Ab (goat anti-rabbitIgG-horseradish peroxidase (HRP), Pierce, Cat. No. 31460) at 1:2000 inassay buffer (PBST-0.5% BSA) and incubated at room temperature for twohours. All incubation and coating steps were performed at roomtemperature on a shaker at 6 RPM. The HRP substrate, TMB(3,3′,5,5″tetramethylbenzidine), used to quantify bound antibody, wasmeasured at 450 nm.

ELISA results are reported as the averages of the triplicate OD (450 nm)value of each sample. Rat GH concentrations were determined by theexceeding mean value plus three times the standard error of the mean(SEM) of the appropriate control value.

The results of the ELISA assays are presented in FIGS. 5-7. FIG. 5presents serum rGH concentrations in BALB/c mice which were dosed bysubcutaneous (SC) injection of 30 μg of non-glycosylated recombinant ratGH. Individual mice sera were tested at a dilution of 1:10, and thegroup average of rGH concentration were reported (n=4 mice per timepoint). Standard error of the mean (SEM) was indicated by the errorbars. As shown in FIG. 5, peak serum concentration of about 240 ng/mlrGH was observed 30 minutes after administration of subcutaneous rGH.

FIG. 6 presents serum rGH concentrations in BALB/c mice which were dosedorally with 100 μg of Delivery Construct 2. Individual mice sera weretested at a dilution of 1:10, and the group average of rGH concentrationwere reported (n=4 mice per time point). Standard error of the mean(SEM) was indicated by the error bars. As shown in FIG. 5, peak serumconcentration of about 280 ng/ml rGH was observed 20 minutes afteradministration of Delivery Construct 2.

FIG. 7 presents a graphical representation comparing pharmacokinetics ofrGH delivered subcutaneously and with Delivery Construct 2. The curvefitting comparison between rGH (SC) and Delivery Construct 2(Oral) wasperformed by evaluating ELISA data as described above using PK Solutions2.0, Pharmacokinetics Data Analysis (Summit Research Services, Montrose,Colo.). As shown in FIG. 7, Delivery Construct 2 yields substantiallyhigher peak serum rGH concentrations than subcutaneous administrationsof rGH. Further, the peak serum concentration is achieved faster withDelivery Construct 2 relative to subcutaneous rGH. The bioavailabilityof rGH delivered with Delivery Construct 2 observed was about 60%relative to rGH administered by subcutaneous injection.

6.8.3. Assays Demonstrating Activity of a Macromolecule FollowingDelivery with a Delivery Construct

This example describes analysis of the biological effects of anexemplary macromolecule, rGH, delivered with Delivery Construct 2 in anin vivo system. In brief, insulin-like growth factor I-binding protein 3(IGF-1-BP3), growth hormone (GH) receptor and insulin-like growth factorI (IGF-I) expression levels were assessed in liver tissue from miceadministered either Delivery Construct 2 or subcutaneous rGH asdescribed above. These transcripts were analyzed because of thewell-characterized effects of GH binding to its receptor on IGF-1-BP3and GH receptor levels. In particular, functional activation of the GHreceptor following binding by GH is known to result in upregulation ofIGF-1-BP3 and downregulation of GH receptor expression. Of these,upregulation of IGF-1-BP3 mRNA expression is believed to be the mostreliable indicator of GH receptor activation. See, e.g., Sondergaard etal., 2003, Am J Physiol Endocrinol Metab 285:E427-32.

Thus, Quantitative Real Time PCR was used to detect and quantify theamount of IGF-1-BP3, GH receptor, IGF-I, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) mRNA in approximately 30 mg of mouse liver tissueprepared as described above. Collected liver tissue was stored at −70°C. until further processing. Real-time detection of PCR was performedusing the Applied Biosystems 7300 Real Time PCR system (AppliedBiosystems, Foster City, Calif.). Total RNA from mouse liver wasisolated according to the RNeasy Protect Mini Kit (Qiagen). Total RNAwas used to generate cDNA for oligo dT oligodeoxynucleotide primer(T12-18) following the protocol for Omniscript Reverse Transcriptase(Qiagen). The primers used to amplify the cDNA were designed usingPrimer Express software (Applied Biosystems), synthesized by Operon(Alameda, Calif.), and are shown in Table 6:

TABLE 6 RT-PCR Primers IGF-I-BP3 (forward): CGCAGAGAAATGGAGGACACA;IGF-I-BP3 (reverse): GGACGCCTCTGGGACTCA; GH receptorGTTGACGAAATAGTGCAACCTGAT; (forward): GH receptor CACGAATCCCGGTCAAACTAA;(reverse): IGF-I (forward): GCTATGGCTCCAGCATTCG; IGF-I (reverse):GCTCCGGAAGCAACACTCA GAPDH (forward): GCAACAGGGTGGTGGACCT GAPDH(reverse): GGATAGGGCCTCTCTTGCTCA

Equal amounts of cDNA were used in duplicate and amplified with the SYBRGreen I Master Mix (Applied Biosystems). The thermal cycling parameterswere as follows: thermal activation for 10 min at 95° C., and 40 cyclesof PCR (melting for 15 s at 95° C. and annealing/extension for 1 min at60° C.). A standard curve was constructed with a dilution curve (1:10,1:100, 1:500, 1:1,000, 1:2,000) of total RNA from a control mouse liversample. A “no template control” was included with each PCR.Amplification efficiencies were validated and normalized against GAPDH.Correct PCR product size was confirmed by electrophoresis through a 1%agarose gel stained with ethidium bromide. Purity of the amplified PCRproducts was determined by a heat-dissociation protocol.

The results of this analysis are shown in FIGS. 8-10. FIG. 8 showsexpression levels of IGF-1-BP3 mRNA in the liver of female BALB/c micetreated with 30 μg recombinant rGH by subcutaneous injection or with 100μg of Delivery Construct 2 by oral gavage. Total RNA extracted from theliver was subjected to quantitative RT-PCR using primers specific forIGF-1-BP3, as described above. Values were normalized toglyceraldehyde-3 phosphate dehydrogenase (GAPDH) and expressed as % ofcontrol. As shown in FIG. 8, administration of subcutaneous rGH resultedin an about 250% increase in IGF-1-BP3 mRNA expression levels in theliver at 60 minutes following administration. In contrast, DeliveryConstruct 2 caused an almost 400% increase in IGF-1-BP3 mRNA expressionlevels in the liver 30 minutes following administration. Thus, oraladministration of Delivery Construct 2 effectively delivered rGH to thebloodstream of the test mice, thereby demonstrating that a deliveryconstruct can effectively deliver an active macromolecule across amucous membrane in an in vivo system. Moreover, Delivery Construct 2delivered more active rGH to the liver than subcutaneous administration,and the effects of administration of rGH were observed substantiallyfaster than possible with subcutaneous administration of rGH.

FIG. 9 shows expression levels of growth hormone (GH) receptor mRNA inthe liver of female B ALB/c mice treated with recombinant rat growthhormone (rGH) by subcutaneous injection (30 μg) or Delivery Construct 2by oral gavage (100 μg). Total RNA extracted from the liver wassubjected to quantitative RT-PCR using primers specific for GH receptor,shown above. Values were normalized to glyceraldehyde-3 phosphatedehydrogenase (GAPDH) and expressed as % of control. As shown in FIG. 9,administration of subcutaneous rGH resulted in a reduction in GHreceptor mRNA expression levels in the liver at 60 minutes followingadministration to about 65% of those observed prior to administration.In contrast, Delivery Construct 2 caused such mRNA levels to decrease toabout 15% of those observed prior to administration. Thus, these resultsconfirm that oral administration of Delivery Construct 2 is effective todeliver rGH to the bloodstream of a subject, and further, that DeliveryConstruct 2 delivers significantly more active rGH to mouse liver thanconventional subcutaneous administration of rGH as shown by the enhanceddownregulation of GH receptor mRNA expression.

FIG. 10 shows expression levels of insulin-like growth factor I (IGF-I)mRNA in the liver of female BALB/c mice treated with recombinant ratgrowth hormone (rGH) by subcutaneous injection (30 μg) or DeliveryConstruct 2 by oral gavage (100 μg). Total RNA extracted from the liverwas subjected to quantitative RT-PCR using primers specific for IGF-I,shown above. Values were normalized to glyceraldehyde-3 phosphatedehydrogenase (GAPDH) and expressed as % of control. As shown in FIG.10, administration of either subcutaneous rGH or Delivery Construct 2resulted in a reduction in IGF-1 mRNA expression levels in the liver at30 minutes following administration to about 20% of those observed priorto administration. Thus, both subcutaneous rGH and orally-administeredDelivery Construct 2 yielded the same effects, demonstrating thatDelivery Construct 2 can deliver active rGH to the bloodstream of a

6.9. Reduced Immunogenicity of Macromolecules Administered with aDelivery Construct

This example shows that an exemplary macromolecule, rGH, administeredorally with Delivery Construct 2, is less immunogenic than rGHadministered subcutaneously.

To assess the relative immunogenicity in mice for rGH administered adelivery construct relative to subcutaneous administration, the serumtiter of anti-rGH IgG antibodies from oral administration of 3, 10, or30 μg Delivery Construct 2 or 3 or 10 μg subcutaneous rGH was determinedin an ELISA assay. To do so, 100 μl 1 ng/μl recombinant rGH diluted incoating buffer (0.2 M NaHCO₃—Na₂CO₃, pH 9.4) was added to Costar EIA/RIAplates, then incubated at room temperature for 16-24 hours. Next, theplates were washed 4 times with 300 μl wash buffer (phosphate-bufferedsaline). The plates were then blocked with 200 μl blocking buffer (0.5%BSA in phosphate-buffered saline) and incubated at room temperature for1 hour. Next, the plates were again washed four times with 300 μl washbuffer.

After preparation of the plates, 100 μl diluted samples, standardpositive control, or assay buffer as negative control was added to theappropriate well and incubated for one hour. Mouse serum samples andpositive control (anti-rGH IgG) were diluted 1:20 in assay buffer (0.5%BSA in phosphate-buffered saline). The plates were then washed fourtimes with 300 μl wash buffer. Next, 100 μl secondary antibody (goatanti-mouse IgG conjugated to horseradish peroxidase, 0.4 mg/ml, Pierce#31430, diluted at 1:6000 in assay buffer and incubated at roomtemperature for one hour. Next, the plates were again washed four timeswith 300 μl wash buffer. Next, 100 μl 3,3′,5,5′-Tetramethylbenzidinesubstrate (Sigma) was added to each well and incubated for 2-10 minutes,depending on color development. 100 μl/well 1M H₂SO₄ was then added tostop the reaction and absorbance read at 450 nm. All assays wereperformed in triplicate and the results averaged.

Representative results of the ELISAs are shown in FIGS. 11A-D. Thegraphs presented in these figures demonstrate that 3, 10, and 30 μgDelivery Construct 2 administered orally elicited a lower titer ofanti-rGH IgG antibodies than either 3 or 10 μg subcutaneous rGH. Inparticular, subcutaneous administration of 10 μg rGH caused asubstantial anti-rGH IgG response in all eight mice, while the eightmice administered 3, 10, or 30 μg Delivery Construct 2 by oral gavagehad minimal anti-rGH IgG responses. Further, these observations wereconsistent whether the sera were diluted 1:25 (FIGS. 11A and 11C) or1:200 (FIGS. 11B and 11D). Finally, it should be noted that each mouseadministered 3, 10, or 30 μg Delivery Construct 2 experienced a minimalimmune response against rGH, as shown by the tight clustering of thedata points in FIGS. 11C and D. Thus, these results demonstrate thatoral administration of Delivery Construct 2 not only delivers moreactive rGH to the liver than possible with subcutaneous injection, butfurther, the active rGH is less immunogenic when administered orallywith Delivery Construct 2 compared to subcutaneous administration.

6.10. Exemplary Delivery Construct for Delivery of Human Growth Hormone

This example describes construction of an exemplary delivery constructfor delivering human growth hormone, termed Delivery Construct 6.Techniques similar to those described in Example 6.1, above, were usedto construct a plasmid used to express Delivery Construct 6. Thenucleotide sequence of the portion of this plasmid that encodes theexemplary delivery construct is presented as FIG. 12, while the aminoacid sequence of the delivery construct is presented as FIG. 13.

6.11. Exemplary Delivery Construct for Delivering Inferferon Alpha

This example describes construction of an exemplary delivery constructfor delivering IFNα (in this case, IFNα-2b), termed Delivery Construct7. Techniques similar to those described in Example 6.1, above, wereused to construct a plasmid used to express Delivery Construct 7. Thenucleotide sequence of the portion of this plasmid that encodes theexemplary Delivery Construct 7 is presented as FIG. 14, while the aminoacid sequence of Delivery Construct 7 is presented as FIG. 15.

6.12. Delivery of Interferon Alpha in an In Vivo System

This example demonstrates the use of Delivery Construct 7 to deliverIFNα-2b to the bloodstream of a subject in a mouse model system.

Using an animal feeding needle, 100 μg of Deli very Construct 7 (in 250μl total volume) was orally administered to female BALB/c mice, 5-6weeks of age (Charles River Laboratories, Wilmington, Mass.). DeliveryConstruct 7 was diluted in 1 mg/ml bovine serum albumin (BSA) andphosphate buffered saline (PBS). At specific times after oral gavage andSC administration, mice were euthanized by CO₂ asphyxiation andexsanguinated. Whole blood was collected and analyzed as describedbelow.

ELISA assays were used to measure serum concentrations of IFNα-2b in onemouse immediately following administration and in three mice at 15, 30,and 75 minutes following administration. The ELISA assays were performedusing R&D Systems Serum ELISA Kit No. 41110-1 according to themanufacturer's instructions.

ELISA results are reported as the averages of the triplicate OD (450 nm)value of each sample. The results of the ELISA assays are presented inFIG. 16. As shown in FIG. 16, IFNα-2b was detected at low (about 3ng/ml) concentration 15 minutes after administration. 30 minutesfollowing administration, serum concentration of IFNα-2b was about 43ng/ml. 45 minutes following administration, serum concentration ofIFNα-2b had fallen to about 13 ng/ml.

6.13. Exemplary Delivery Construct for Delivering Proinsulin

This example describes construction of an exemplary delivery constructfor delivering proinsulin, termed Delivery Construct 8. Techniquessimilar to those described in Example 6.1, above, are used to constructa plasmid used to express Delivery Construct 8. The amino acid sequenceof Delivery Construct 8 is presented as FIG. 17.

6.14. Exemplary Delivery Construct for Delivering Insulin

This example describes construction of an exemplary delivery constructfor delivering insulin, termed Delivery Construct 9. Techniques similarto those described in Example 6.1, above, are used to construct aplasmid used to express Delivery Construct 9, with certainmodifications.

In particular, the scheme used to express Delivery Construct 9 ismodified because insulin comprises two separate amino acid chains. Inthis example, the B-chain of insulin is expressed together with theremainder of the Delivery Construct, constructed according to thegeneral scheme presented in Example 6.1. Amino acids corresponding tothe A-chain are made either synthetically (e.g., chemically synthesizingthe A-chain peptide from amino acids) or recombinantly (e.g., expressedin a suitable recombinant system such as, for example, E. coli, yeast,etc.) The two polypeptides are then combined under conditions thatpermit association of the A-chain and B-chain. Then, disulfide bonds aremade between the two chains of insulin as found in native insulin byapplication of mildly oxidizing conditions. The amino acid sequence ofthe two amino acid chains of Delivery Construct 9 is presented as FIG.17.

The present invention provides, inter alia, delivery constructs andmethods of inducing an immune response in a subject. While many specificexamples have been provided, the above description is intended toillustrate rather than limit the invention. Many variations of theinvention will become apparent to those skilled in the art upon reviewof this specification. The scope of the invention should, therefore, bedetermined not with reference to the above description, but insteadshould be determined with reference to the appended claims along withtheir full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. Citation of these documents is not an admissionthat any particular reference is “prior art” to this invention.

TABLE 7 Human Peptidases by Class Aspartic-type peptidases BAE1_HUMAN(P56817) BAE2_HUMAN (Q9Y5Z0) CATD_HUMAN (P07339) CATE_HUMAN (P14091)NAP1_HUMAN (O96009) PEPA_HUMAN (P00790 PEPC_HUMAN (P20142) RENI_HUMAN(P00797) VPRT_HUMAN (P10265) Other Peptidases FAC2_HUMAN (Q9Y256)Cysteine-type peptidases BLMH_HUMAN (Q13867) CATB_HUMAN (P07858)CATC_HUMAN (P53634) CATF_HUMAN (Q9UBX1) CATH_HUMAN (P09668) CATK_HUMAN(P43235) CATL_HUMAN (P07711) CATO_HUMAN (P43234) CATS_HUMAN (P25774)CATW_HUMAN (P56202) CATZ_HUMAN (Q9UBR2) CSL2_HUMAN (O60911) TNAG_HUMAN(Q9UJW2) CAN1_HUMAN (P07384) CAN2_HUMAN (P17655) CAN3_HUMAN (P20807)CAN5_HUMAN (O15484) CAN6_HUMAN (Q9Y6Q1) CAN7_HUMAN (Q9Y6W3) CAN9_HUMAN(O14815) CANA_HUMAN (Q9HC96) CANB_HUMAN (Q9UMQ6) UBL1_HUMAN (P09936)UBL3_HUMAN (P15374) UBL5_HUMAN (Q9Y5K5) GPI8_HUMAN (Q92643) LGMN_HUMAN(Q99538) CFLA_HUMAN (O15519) I1BC_HUMAN (P29466) ICE2_HUMAN (P42575)ICE3_HUMAN (P42574) ICE4_HUMAN (P49662) ICE5_HUMAN (P51878) ICE6_HUMAN(P55212) ICE7_HUMAN (P55210) ICE8_HUMAN (Q14790) ICE9_HUMAN (P55211)ICEA_HUMAN (Q92851) ICEE_HUMAN (P31944) MLT1_HUMAN (Q9UDY8) PGPI_HUMAN(Q9NXJ5) FAFX_HUMAN (Q93008) FAFY_HUMAN (O00507) UB10_HUMAN (Q14694)UB11_HUMAN (P51784) UB12_HUMAN (O75317) UB13_HUMAN (Q92995) UB14_HUMAN(P54578) UB15_HUMAN (Q9Y4E8) UB16_HUMAN (Q9Y5T5) UB18_HUMAN (Q9UMW8)UB19_HUMAN (O94966) UB20_HUMAN (Q9Y2K6) UB21_HUMAN (Q9UK80) UB22_HUMAN(Q9UPT9) UB24_HUMAN (Q9UPU5) UB25_HUMAN (Q9UHP3) UB26_HUMAN (Q9BXU7)UB28_HUMAN (Q96RU2) UB29_HUMAN (Q9HBJ7) UB32_HUMAN (Q8NFA0) UB33_HUMAN(Q8TEY7) UB35_HUMAN (Q9P2H5) UB36_HUMAN (Q9P275) UB37_HUMAN (Q86T82)UB38_HUMAN (Q8NB14) UB40_HUMAN (Q9NVE5) UB42_HUMAN (Q9H9J4) UB44_HUMAN(Q9H0E7) UB46_HUMAN (P62068) UBP1_HUMAN (O94782) UBP2_HUMAN (O75604)UBP3_HUMAN (Q9Y6I4) UBP4_HUMAN (Q13107) UBP5_HUMAN (P45974) UBP6_HUMAN(P35125) UBP7_HUMAN (Q93009) UBP8_HUMAN (P40818) GGH_HUMAN (Q92820)SEN1_HUMAN (Q9P0U3) SEN3_HUMAN (Q9H4L4) SEN5_HUMAN (Q96HI0) SEN6_HUMAN(Q9GZR1) SEN7_HUMAN (Q9BQF6) SEN8_HUMAN (Q96LD8) SNP2_HUMAN (Q9HC62)ESP1_HUMAN (Q14674) Metallopeptidases AMPB_HUMAN (Q9H4A4) AMPE_HUMAN(Q07075) AMPN_HUMAN (P15144) ART1_HUMAN (Q9NZ08) LCAP_HUMAN (Q9UIQ6)LKHA_HUMAN (P09960) PSA_HUMAN (P55786) RNPL_HUMAN (Q9HAU8) THDE_HUMAN(Q9UKU6) ACET_HUMAN (P22966) ACE_HUMAN (P12821) MEPD_HUMAN (P52888)NEUL_HUMAN (Q9BYT8) PMIP_HUMAN (Q99797) MM01_HUMAN (P03956) MM02_HUMAN(P08253) MM03_HUMAN (P08254) MM07_HUMAN (P09237) MM08_HUMAN (P22894)MM09_HUMAN (P14780) MM10_HUMAN (P09238) MM11_HUMAN (P24347) MM12_HUMAN(P39900) MM13_HUMAN (P45452) MM14_HUMAN (P50281) MM15_HUMAN (P51511)MM16_HUMAN (P51512) MM17_HUMAN (Q9ULZ9) MM19_HUMAN (Q99542) MM20_HUMAN(O60882) MM21_HUMAN (Q8N119) MM24_HUMAN (Q9Y5R2) MM25_HUMAN (Q9NPA2)MM26_HUMAN (Q9NRE1) MM28_HUMAN (Q9H239) BMP1_HUMAN (P13497) MEPA_HUMAN(Q16819) MEPB_HUMAN (Q16820) AD02_HUMAN (Q99965) AD07_HUMAN (Q9H2U9)AD08_HUMAN (P78325) AD09_HUMAN (Q13443) AD10_HUMAN (O14672) AD11_HUMAN(O75078) AD12_HUMAN (O43184) AD15_HUMAN (Q13444) AD17_HUMAN (P78536)AD18_HUMAN (Q9Y3Q7) AD19_HUMAN (Q9H013) AD20_HUMAN (O43506) AD21_HUMAN(Q9UKJ8) AD22_HUMAN (Q9P0K1) AD28_HUMAN (Q9UKQ2) AD29_HUMAN (Q9UKF5)AD30_HUMAN (Q9UKF2) AD33_HUMAN (Q9BZ11) AT10_HUMAN (Q9H324) AT12_HUMAN(P58397) AT14_HUMAN (Q8WXS8) AT15_HUMAN (Q8TE58) AT16_HUMAN (Q8TE57)AT17_HUMAN (Q8TE56) AT18_HUMAN (Q8TE60) AT19_HUMAN (Q8TE59) AT20_HUMAN(P59510) ATS1_HUMAN (Q9UHI8) ATS2_HUMAN (O95450) ATS3_HUMAN (O15072)ATS4_HUMAN (O75173) ATS5_HUMAN (Q9UNA0) ATS6_HUMAN (Q9UKP5) ATS7_HUMAN(Q9UKP4) ATS8_HUMAN (Q9UP79) ATS9_HUMAN (Q9P2N4) ECE1_HUMAN (P42892)ECE2_HUMAN (O60344) ECEL_HUMAN (O95672) KELL_HUMAN (P23276) NEP_HUMAN(P08473) PEX_HUMAN (P78562) CBP1_HUMAN (P15085) CBP2_HUMAN (P48052)CBP4_HUMAN (Q9UI42) CBP5_HUMAN (Q8WXQ8) CBP6_HUMAN (Q8N4T0) CBPB_HUMAN(P15086) CBPC_HUMAN (P15088) CBPD_HUMAN (O75976) CBPE_HUMAN (P16870)CBPM_HUMAN (P14384) CBPN_HUMAN (P15169) CPX2_HUMAN (Q8N436) CPXM_HUMAN(Q96SM3) IDE_HUMAN (P14735) MPPA_HUMAN (Q10713) MPPB_HUMAN (O75439)NRDC_HUMAN (O43847) UCR1_HUMAN (P31930) UCR2_HUMAN (P22695) AMPL_HUMAN(P28838) PEL1_HUMAN (Q8NDH3) DNPE_HUMAN (Q9ULA0) MDP1_HUMAN (P16444)CGL1_HUMAN (Q96KP4) CGL2_HUMAN (Q96KN2) ACY1_HUMAN (Q03154) GCP_HUMAN(Q9NPF4) AMP1_HUMAN (P53582) PEPD_HUMAN (P12955) XPP2_HUMAN (O43895)AMP2_HUMAN (P50579) P2G4_HUMAN (Q9UQ80) FOH1_HUMAN (Q04609) NLD2_HUMAN(Q9Y3Q0) NLDL_HUMAN (Q9UQQ1) TFR1_HUMAN (P02786) TFR2_HUMAN (Q9UP52)AF31_HUMAN (O43931) AF32_HUMAN (Q9Y4W6) SPG7_HUMAN (Q9UQ90) YME1_HUMAN(Q96TA2) PAPA_HUMAN (Q13219) FAC1_HUMAN (O75844) DPP3_HUMAN (Q9NY33)MS2P_HUMAN (O43462) Serine-type peptidases ACRL_HUMAN (P58840)ACRO_HUMAN (P10323) APOA_HUMAN (P08519) BSS4_HUMAN (Q9GZN4) C1R_HUMAN(P00736) C1S_HUMAN (P09871) CAP7_HUMAN (P20160) CATG_HUMAN (P08311)CFAB_HUMAN (P00751) CFAD_HUMAN (P00746) CFAI_HUMAN (P05156) CLCR_HUMAN(Q99895) CO2_HUMAN (P06681) CORI_HUMAN (Q9Y5Q5) CRAR_HUMAN (P48740)CTRB_HUMAN (P17538) CTRL_HUMAN (P40313) DES1_HUMAN (Q9UL52) EL1_HUMAN(Q9UNI1) EL2A_HUMAN (P08217) EL2B_HUMAN (P08218) EL3A_HUMAN (P09093)EL3B_HUMAN (P08861) ELNE_HUMAN (P08246) ENTK_HUMAN (P98073) FA10_HUMAN(P00742) FA11_HUMAN (P03951) FA12_HUMAN (P00748) FA7_HUMAN (P08709)FA9_HUMAN (P00740) GRAA_HUMAN (P12544) GRAB_HUMAN (P10144) GRAH_HUMAN(P20718) GRAK_HUMAN (P49863) GRAM_HUMAN (P51124) HATT_HUMAN (O60235)HEPS_HUMAN (P05981) HGFA_HUMAN (Q04756) HGFL_HUMAN (P26927) HGF_HUMAN(P14210) HPTR_HUMAN (P00739) HPT_HUMAN (P00738) KAL_HUMAN (P03952)KLK1_HUMAN (P06870) KLK2_HUMAN (P20151) KLK3_HUMAN (P07288) KLK4_HUMAN(Q9Y5K2) KLK5_HUMAN (Q9Y337) KLK6_HUMAN (Q92876) KLK7_HUMAN (P49862)KLK8_HUMAN (O60259) KLK9_HUMAN (Q9UKQ9) KLKA_HUMAN (O43240) KLKB_HUMAN(Q9UBX7) KLKC_HUMAN (Q9UKR0) KLKD_HUMAN (Q9UKR3) KLKE_HUMAN (Q9P0G3)KLKF_HUMAN (Q9H2R5) LCLP_HUMAN (P34168) MAS2_HUMAN (O00187) MCT1_HUMAN(P23946) NETR_HUMAN (P56730) PLMN_HUMAN (P00747) PR27_HUMAN (Q9BQR3)PRN3_HUMAN (P24158) PRTC_HUMAN (P04070) PRTZ_HUMAN (P22891) PS23_HUMAN(O95084) PSS8_HUMAN (Q16651) ST14_HUMAN (Q9Y5Y6) TEST_HUMAN (Q9Y6M0)THRB_HUMAN (P00734) TMS2_HUMAN (O15393) TMS3_HUMAN (P57727) TMS4_HUMAN(Q9NRS4) TMS5_HUMAN (Q9H3S3) TMS6_HUMAN (Q8IU80) TPA_HUMAN (P00750)TRB1_HUMAN (Q15661) TRB2_HUMAN (P20231) TRY1_HUMAN (P07477) TRY2_HUMAN(P07478) TRY3_HUMAN (P35030) TRYA_HUMAN (P15157) TRYD_HUMAN (Q9BZJ3)TRYG_HUMAN (Q9NRR2) TS50_HUMAN (Q9UI38) UROK_HUMAN (P00749) HRA1_HUMAN(Q92743) HRA2_HUMAN (O43464) HRA3_HUMAN (P83110) HRA4_HUMAN (P83105)FURI_HUMAN (P09958) MS1P_HUMAN (Q14703) NEC1_HUMAN (P29120) NEC2_HUMAN(P16519) PCK5_HUMAN (Q92824) PCK6_HUMAN (P29122) PCK7_HUMAN (Q16549)PCK9_HUMAN (Q8NBP7) TPP2_HUMAN (P29144) PPCE_HUMAN (P48147) DPP4_HUMAN(P27487) DPP6_HUMAN (P42658) SEPR_HUMAN (Q12884) ACPH_HUMAN (P13798)CPVL_HUMAN (Q9H3G5) PRTP_HUMAN (P10619) RISC_HUMAN (Q9HB40) CLPP_HUMAN(Q16740) LONM_HUMAN (P36776) SPC3_HUMAN (Q9BY50) SPC4_HUMAN (P21378)DPP2_HUMAN (Q9UHL4) PCP_HUMAN (P42785) TSSP_HUMAN (Q9NQE7) HYEP_HUMAN(P07099) TPP1_HUMAN (O14773) RHB1_HUMAN (O75783) RHB2_HUMAN (Q9NX52)RHB4_HUMAN (P58872) Threonine-type peptidases PS7L_HUMAN (Q8TAA3)PSA1_HUMAN (P25786) PSA2_HUMAN (P25787) PSA3_HUMAN (P25788) PSA4_HUMAN(P25789) PSA5_HUMAN (P28066) PSA6_HUMAN (P60900) PSA7_HUMAN (O14818)PSB1_HUMAN (P20618) PSB2_HUMAN (P49721) PSB3_HUMAN (P49720) PSB4_HUMAN(P28070) PSB5_HUMAN (P28074) PSB6_HUMAN (P28072) PSB7_HUMAN (Q99436)PSB8_HUMAN (P28062) PSB9_HUMAN (P28065) PSBA_HUMAN (P40306)

1. A delivery construct, comprising: a)- a receptor binding domain, b)-a transcytosis domain, c)- a macromolecule to be delivered to a subject,and d)- a cleavable linker, wherein cleavage at said cleavable linkerseparates said macromolecule from the remainder of said construct, andwherein said cleavable linker is cleavable by an enzyme that i) exhibitsgreater activity at a basal-lateral membrane of a polarized epithelialcell of said subject than at an apical membrane of the polarizedepithelial cell, or ii) exhibits greater activity in the plasma of saidsubject than at an apical membrane of the polarized epithelial cell ofthe subject.
 2. The delivery construct of claim 1, further comprising asecond cleavable linker.
 3. The delivery construct of claim 1, whereinsaid cleavable linker comprises an amino acid sequence that is selectedfrom the group consisting of Ala-Ala-Pro-Phe (SEQ ID NO.:4), Gly-Gly-Phe(SEQ ID NO.:5), Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu (SEQ IDNO.:7), Ala-Ala-Leu (SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9),Val-Gly-Arg (SEQ ID NO.:10).
 4. The delivery construct of claim 1,wherein said enzyme that is present at a basal-lateral membrane of apolarized epithelial cell is selected from the group consisting ofCathepsin GI, Chymotrypsin I, Elastase I, Subtilisin AI, Subtilisin AII,Thrombin I, and Urokinase I.
 5. The delivery construct of claim 1,wherein said receptor binding domain is selected from the groupconsisting of receptor binding domains from Pseudomonas exotoxin A,cholera toxin, botulinum toxin, diptheria toxin, shiga toxin, orshiga-like toxin; monoclonal antibodies; polyclonal antibodies;single-chain antibodies; TGF α; EGF; IGF-I; IGF-II; IGF-III; IL-1; IL-2;IL-3; IL-6; MIP-1a; MIP-1b; MCAF; and IL-8.
 6. The delivery construct ofclaim 1, wherein said receptor binding domain binds to a cell-surfacereceptor that is selected from the group consisting of α2-macroglobulinreceptor, epidermal growth factor receptor, transferrin receptor,chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNFα receptor, TOLLreceptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, and VEGFreceptor.
 7. The delivery construct of claim 5, wherein said receptorbinding domain of Pseudomonas exotoxin A is Domain Ia of Pseudomonasexotoxin A.
 8. The delivery construct of claim 7, wherein said receptorbinding domain of Pseudomonas exotoxin A has an amino acid sequence thatis SEQ ID NO.:1.
 9. The delivery construct of claim 1, wherein saidtranscytosis domain is selected from the group consisting oftranscytosis domains from Pseudomonas exotoxin A, botulinum toxin,diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. colienterotoxin, shiga toxin, and shiga-like toxin.
 10. The deliveryconstruct of claim 9, wherein said transcytosis domain is Pseudomonasexotoxin A transcytosis domain.
 11. The delivery construct of claim 10,wherein said Pseudomonas exotoxin A transcytosis domain has an aminoacid sequence that is SEQ ID NO.:2.
 12. The delivery construct of claim1, wherein said macromolecule is selected from the group of a nucleicacid, a peptide, a polypeptide, a protein, and a lipid.
 13. The deliveryconstruct of claim 12, wherein said polypeptide is selected from thegroup consisting of polypeptide hormones, cytokines, chemokines, growthfactors, and clotting factors.
 14. The delivery construct of claim 13,wherein said polypeptide is selected from the group consisting of IGF-I,IGF-II, IGF-III, EGF, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, IL-1, IL-2,IL-3, IL-6, IL-8, IL-12, EPO, growth hormone, factor VII, vasopressin,calcitonin, parathyroid hormone, luteinizing hormone-releasing factor,tissue plasminogen activators, proinsulin, insulin, glucocorticoid,amylin, adrenocorticototropin, enkephalin, and glucagon-like peptide 1.15. The delivery construct of claim 14, wherein said polypeptide ishuman growth hormone.
 16. The delivery construct of claim 12, whereinsaid protein is human insulin.
 17. The delivery construct of claim 12,wherein said protein is human IFN-α.
 18. The delivery construct of claim12, wherein said protein is human IFN-α2b.
 19. The delivery construct ofclaim 12, wherein said protein is human proinsulin.
 20. The deliveryconstruct of claim 12, further comprising a second macromolecule that isselected from the group of a nucleic acid, a peptide, a polypeptide, aprotein, a lipid, or a small organic molecule and a second cleavablelinker, wherein cleavage at said second cleavable linker separates saidsecond macromolecule from the remainder of said construct.
 21. Thedelivery construct of claim 17, wherein said macromolecule is a firstpolypeptide and said second macromolecule is a second polypeptide. 22.The delivery construct of claim 21, wherein said first polypeptide andsaid second polypeptide associate to form a multimer.
 23. The deliveryconstruct of claim 22, wherein said multimer is a dimer, tetramer, oroctamer.
 24. The delivery construct of claim 23, wherein said dimer isan antibody.
 25. A polynucleotide that encodes a delivery construct,said delivery construct comprising: a)- a receptor binding domain, b)- atranscytosis domain, c)- a macromolecule to be delivered to a subject,and d)- a cleavable linker, wherein cleavage at said cleavable linkerseparates said macromolecule from the remainder of said construct, andwherein said cleavable linker is cleavable by an enzyme that i) exhibitsgreater activity at a basal-lateral membrane of a polarized epithelialcell of said subject than at an apical membrane of the polarizedepithelial cell, or ii) exhibits greater activity in the plasma of saidsubject than at an apical membrane of the polarized epithelial cell ofthe subject.
 26. A polynucleotide that hybridizes under stringenthybridization conditions to the polynucleotide of claim
 25. 27. Thepolynucleotide of claim 25, wherein said delivery construct furthercomprises a second cleavable linker.
 28. The polynucleotide of claim 25,wherein said cleavable linker comprises an amino acid sequence that isselected from the group consisting of Ala-Ala-Pro-Phe (SEQ ED NO.:4),Gly-Gly-Phe (SEQ ID NO.:5), Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu(SEQ ID NO.:7), Ala-Ala-Leu (SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9),Val-Gly-Arg (SEQ ID NO.:10).
 29. The polynucleotide of claim 25, whereinsaid enzyme that is present at a basal-lateral membrane of a polarizedepithelial cell is selected from the group consisting of Cathepsin GI,Chymotrypsin I, Elastase I, Subtilisin AI, Subtilisin AII, Thrombin I,and Urokinase I.
 30. The polynucleotide of claim 25, wherein saidreceptor binding domain is selected from the group consisting ofreceptor binding domains from Pseudomonas exotoxin A, cholera toxin,botulinum toxin, diptheria toxin, shiga toxin, or shiga-like toxin;monoclonal antibodies; polyclonal antibodies; single-chain antibodies;TGF α; EGF; IGF-I; IGF-II; IGF-III; IL-1; IL-2; IL-3; IL-6; MIP-1a;MIP-1b; MCAF; and IL-8.
 31. The polynucleotide of claim 25, wherein saidreceptor binding domain binds to a cell-surface receptor that isselected from the group consisting of α2-macroglobulin receptor, EGFR,IGFR, transferrin receptor, chemokine receptor, CD25, CD11B, CD11C,CD80, CD86, TNFα receptor, TOLL receptor, M-CSF receptor, GM-CSFreceptor, scavenger receptor, and VEGF receptor.
 32. The polynucleotideof claim 30, wherein said receptor binding domain of Pseudomonasexotoxin A is Domain Ia of Pseudomonas exotoxin A.
 33. Thepolynucleotide of claim 31, wherein said receptor binding domain ofPseudomonas exotoxin A has an amino acid sequence that is SEQ ID NO.:1.34. The polynucleotide of claim 25, wherein said transcytosis domain isselected from the group consisting of transcytosis domains fromPseudomonas exotoxin A, botulinum toxin, diptheria toxin, pertussistoxin, cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, andshiga-like toxin.
 35. The polynucleotide of claim 34, wherein saidtranscytosis domain is Pseudomonas exotoxin A transcytosis domain. 36.The polynucleotide of claim 35, wherein said Pseudomonas exotoxin Atranscytosis domain has an amino acid sequence that is SEQ ID NO.: 2.37. The polynucleotide of claim 25, wherein said macromolecule isselected from the group of a peptide, a polypeptide, and a protein. 38.The polynucleotide of claim 37, wherein said polypeptide is selectedfrom the group consisting of polypeptide hormones, cytokines,chemokines, growth factors, and clotting factors.
 39. The polynucleotideof claim 38, wherein said polypeptide is selected from the groupconsisting of IGF-I, IGF-II, IGF-1H, EGF, IFN-α, IFN-α, 2b, IFN-β,IFN-γ, G-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-6, IL-8, IL-12, EPO, growthhormone, factor VII, vasopressin, calcitonin, parathyroid hormone,luteinizing hormone-releasing factor, tissue plasminogen activators,proinsulin, insulin, glucocorticoid, amylin, adrenocorticototropin,enkephalin, and glucagon-like peptide
 1. 40. The polynucleotide of claim39, wherein said polypeptide is human growth hormone.
 41. Thepolynucleotide of claim 39, wherein said protein is human insulin.
 42. Apolynucleotide that encodes a delivery construct, said polynucleotidecomprising: a)- a nucleic acid sequence encoding a receptor bindingdomain, b)- a nucleic acid sequence encoding a transcytosis domain, c)-a nucleic acid sequence encoding a cleavable linker, and d)- a nucleicacid sequence comprising a polylinker insertion site, wherein cleavageat said cleavable linker separates said macromolecule from the remainderof said construct, and wherein said cleavable linker is cleavable by anenzyme that i) exhibits greater activity at a basal-lateral membrane ofa polarized epithelial cell of said subject than at an apical membraneof the polarized epithelial cell, or ii) exhibits greater activity inthe plasma of said subject than at an apical membrane of the polarizedepithelial cell of the subject.
 43. An expression vector comprising thepolynucleotide of claim
 25. 44. A cell comprising the expression vectorof claim
 43. 45. A composition comprising a delivery construct, saiddelivery construct comprising: a)- a receptor binding domain, b)- atranscytosis domain, c)- a macromolecule to be delivered to a subject,and d)- a cleavable linker, wherein cleavage at said cleavable linkerseparates said macromolecule from the remainder of said construct, andwherein said cleavable linker is cleavable by an enzyme that i) exhibitsgreater activity at a basal-lateral membrane of a polarized epithelialcell of said subject than at an apical membrane of the polarizedepithelial cell, or ii) exhibits greater activity in the plasma of saidsubject than at an apical membrane of the polarized epithelial cell ofthe subject.
 46. The composition of claim 45, wherein said compositionfurther comprises a pharmaceutically acceptable diluent, excipient,vehicle, or carrier.
 47. The composition of claim 45, wherein saidcomposition is formulated for nasal or oral administration.
 48. A methodfor delivering a macromolecule to a subject, comprising contacting anapical surface of a polarized epithelial cell of the subject with adelivery construct, wherein said delivery construct comprises a receptorbinding domain, a transcytosis domain, a cleavable linker, and themacromolecule, wherein the transcytosis domain transcytosis themacromolecule to and through the basal-lateral membrane of saidepithelial cell, wherein cleavage at said cleavable linker separatessaid macromolecule from the remainder of said construct, and whereinsaid cleavable linker is cleavable by an enzyme that i) exhibits greateractivity at a basal-lateral membrane of a polarized epithelial cell ofsaid subject than at an apical membrane of the polarized epithelialcell, or ii) exhibits greater activity in the plasma of said subjectthan at an apical membrane of the polarized epithelial cell of thesubject, and wherein cleavage at the cleavable linker separates themacromolecule from the remainder of the delivery construct, therebydelivering the macromolecule to the subject.
 49. The method of claim 48,wherein said receptor binding domain is selected from the groupconsisting of receptor binding domains from Pseudomonas exotoxin A,cholera toxin, diptheria toxin, shiga toxin, or shiga-like toxin;monoclonal antibodies; polyclonal antibodies; single-chain antibodies;TGF α; EGF; IGF-I; IGF-II; IGF-III; IL-1; IL-2; IL-3; IL-6; MIP-1a;MIP-1b; MCAF; and IL-8.
 50. The method of claim 48, wherein saidreceptor binding domain binds to a cell surface receptor selected fromthe group consisting of α2-macroglobulin receptor, EGFR, IGFR,transferrin receptor, chemokine receptor, CD25, CD11B, CD11C, CD80,CD86, TNFα receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor,scavenger receptor, and VEGF receptor.
 51. The method of claim 48,wherein said transcytosis domain is selected from the group consistingof transcytosis domains from Pseudomonas exotoxin A, botulinum toxin,diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. colienterotoxin, shiga toxin, and shiga-like toxin.
 52. The method of claim48, wherein said macromolecule is selected from the group consisting ofa peptide, a polypeptide, a protein, a nucleic acid, and a lipid. 53.The method of claim 48, wherein said enzyme that is present at abasal-lateral membrane of a polarized epithelial cell is selected fromthe group consisting of Cathepsin GI, Chymotrypsin I, Elastase I,Subtilisin AI, Subtilisin AII, Thrombin I, and Urokinase I.
 54. Themethod of claim 48, wherein said cleavable linker comprises an aminoacid sequence that is selected from the group consisting ofAla-Ala-Pro-Phe (SEQ ID NO.:4), Gly-Gly-Phe (SEQ ID NO.:5),Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu (SEQ ID NO.:7), Ala-Ala-Leu(SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9), Val-Gly-Arg (SEQ ID NO.:10).
 55. The method of claim 48, wherein the epithelial cell is selectedfrom the group consisting of nasal epithelial cells, oral epithelialcells, intestinal epithelial cells, rectal epithelial cells, vaginalepithelial cells, and pulmonary epithelial cells.
 56. The method ofclaim 48, wherein said mammal is a human.
 57. The method of claim 48,wherein said delivery construct contacts the apical membrane of theepithelial cell.
 58. A method for delivering a macromolecule to thebloodstream of a subject, comprising contacting the delivery constructof claim 1 to an apical surface of a polarized epithelial cell of thesubject, such that the macromolecule is delivered to the bloodstream ofthe subject, wherein a lower titer of antibodies specific for themacromolecule is induced in the serum of the subject than is induced bysubcutaneously administering the macromolecule to a subject separatelyfrom the remainder of the delivery construct.
 59. The method of claim58, wherein the macromolecule is selected from the group consisting of apeptide, a polypeptide, a protein, a nucleic acid, and a lipid.
 60. Themethod of claim 58, wherein the macromolecule is selected from the groupconsisting of polypeptide hormones, cytokines, chemokines, growthfactors, and clotting factors.
 61. The method of claim 58, wherein themacromolecule is selected from the group consisting of IGF-I, IGF-II,IGF-III, EGF, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, IL-1, IL-2, IL-3,IL-6, IL-8, IL-12, EPO, growth hormone, factor VII, vasopressin,calcitonin, parathyroid hormone, luteinizing hormone-releasing factor,tissue plasminogen activators, proinsulin, insulin, glucocorticoid,amylin, adrenocorticototropin, enkephalin, and glucagon-like peptide 1.62. The method of claim 58, wherein the macromolecule is human growthhormone.
 63. The method of claim 58, wherein the macromolecule is humaninsulin.
 64. The method of claim 58, wherein the subject is a mouse,dog, goat, or human.
 65. The method of claim 58, wherein the titer ofantibodies specific for the macromolecule induced in the serum of thesubject by the macromolecule delivered by with the delivery construct isless than about 75% of the titer of antibodies induced by subcutaneouslyadministering the macromolecule to a subject separately from theremainder of the delivery construct.
 66. The method of claim 58, whereinthe titer of antibodies specific for the macromolecule induced in theserum of the subject by the macromolecule delivered by the deliveryconstruct is less than about 50% of the titer of antibodies induced bysubcutaneously administering the macromolecule to a subject separatelyfrom the remainder of the delivery construct.
 67. The method of claim58, wherein the titer of antibodies specific for the macromoleculeinduced in the serum of the subject by the macromolecule delivered bythe delivery construct is less than about 25% of the titer of antibodiesinduced by subcutaneously administering the macromolecule to a subjectseparately from the remainder of the delivery construct.
 68. The methodof claim 58, wherein the titer of antibodies specific for themacromolecule induced in the serum of the subject by the macromoleculedelivered by the delivery construct is less than about 10% of the titerof antibodies induced by subcutaneously administering the macromoleculeto a subject separately from the remainder of the delivery construct.